Novel cyclic cationic lipids and methods of use

ABSTRACT

The present invention provides compositions and methods for the delivery of therapeutic agents to cells. In particular, these include novel cationic lipids and nucleic acid-lipid particles that provide efficient encapsulation of nucleic acids and efficient delivery of the encapsulated nucleic acid to cells in vivo. The compositions of the present invention are highly potent, thereby allowing effective knock-down of a specific target protein at relatively low doses. In addition, the compositions and methods of the present invention are less toxic and provide a greater therapeutic index compared to compositions and methods previously known in the art.

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims priority to U.S. Provisional ApplicationNo. 61/334,096, filed May 12, 2010, the disclosure of which is herebyincorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

Therapeutic nucleic acids include, e.g., small interfering RNA (siRNA),microRNA (miRNA), antisense oligonucleotides, ribozymes, plasmids, andimmune-stimulating nucleic acids. These nucleic acids act via a varietyof mechanisms. In the case of interfering RNA molecules such as siRNAand miRNA, these nucleic acids can down-regulate intracellular levels ofspecific proteins through a process termed RNA interference (RNAi).Following introduction of interfering RNA into the cell cytoplasm, thesedouble-stranded RNA constructs can bind to a protein termed RISC. Thesense strand of the interfering RNA is displaced from the RISC complex,providing a template within RISC that can recognize and bind mRNA with acomplementary sequence to that of the bound interfering RNA. Havingbound the complementary mRNA, the RISC complex cleaves the mRNA andreleases the cleaved strands. RNAi can provide down-regulation ofspecific proteins by targeting specific destruction of the correspondingmRNA that encodes for protein synthesis.

The therapeutic applications of RNAi are extremely broad, sinceinterfering RNA constructs can be synthesized with any nucleotidesequence directed against a target protein.

To date, siRNA constructs have shown the ability to specificallydown-regulate target proteins in both in vitro and in vivo models. Inaddition, siRNA constructs are currently being evaluated in clinicalstudies.

However, two problems currently faced by interfering RNA constructs are,first, their susceptibility to nuclease digestion in plasma and, second,their limited ability to gain access to the intracellular compartmentwhere they can bind RISC when administered systemically as freeinterfering RNA molecules. These double-stranded constructs can bestabilized by the incorporation of chemically modified nucleotidelinkers within the molecule, e.g., phosphothioate groups. However, suchchemically modified linkers provide only limited protection fromnuclease digestion and may decrease the activity of the construct.Intracellular delivery of interfering RNA can be facilitated by the useof carrier systems such as polymers, cationic liposomes, or by thecovalent attachment of a cholesterol moiety to the molecule. However,improved delivery systems are required to increase the potency ofinterfering RNA molecules such as siRNA and miRNA and to reduce oreliminate the requirement for chemically modified nucleotide linkers.

In addition, problems remain with the limited ability of therapeuticnucleic acids such as interfering RNA to cross cellular membranes (see,Vlassov et al., Biochim. Biophys. Acta, 1197:95-1082 (1994)) and in theproblems associated with systemic toxicity, such as complement-mediatedanaphylaxis, altered coagulatory properties, and cytopenia (Galbraith etal., Antisense Nucl. Acid Drug Des., 4:201-206 (1994)).

To attempt to improve efficacy, investigators have also employedlipid-based carrier systems to deliver chemically modified or unmodifiedtherapeutic nucleic acids. Zelphati et al. (J. Contr. Rel., 41:99-119(1996)) describes the use of anionic (conventional) liposomes, pHsensitive liposomes, immunoliposomes, fusogenic liposomes, and cationiclipid/antisense aggregates. Similarly, siRNA has been administeredsystemically in cationic liposomes, and these nucleic acid-lipidparticles have been reported to provide improved down-regulation oftarget proteins in mammals including non-human primates (Zimmermann etal., Nature, 441: 111-114 (2006)).

In spite of this progress, there remains a need in the art for improvedlipid-therapeutic nucleic acid compositions that are suitable forgeneral therapeutic use. Preferably, these compositions wouldencapsulate nucleic acids with high efficiency, have high drug:lipidratios, protect the encapsulated nucleic acid from degradation andclearance in serum, be suitable for systemic delivery, and provideintracellular delivery of the encapsulated nucleic acid. In addition,these nucleic acid-lipid particles should be well-tolerated and providean adequate therapeutic index, such that patient treatment at aneffective dose of the nucleic acid is not associated with significanttoxicity and/or risk to the patient. The present invention provides suchcompositions, methods of making the compositions, and methods of usingthe compositions to introduce nucleic acids into cells, including forthe treatment of diseases.

BRIEF SUMMARY OF THE INVENTION

The present invention provides novel cationic (amino) lipids and lipidparticles comprising these lipids, which are advantageous for the invivo delivery of active agents or therapeutic agents such as nucleicacids, as well as lipid particles such as nucleic acid-lipid particlecompositions suitable for in vivo therapeutic use. The present inventionalso provides methods of making these lipid compositions, as well asmethods of introducing active agents or therapeutic agents such asnucleic acids into cells using these lipid compositions, e.g., for thetreatment of various disease conditions.

In one aspect, the present invention provides a cationic lipid ofFormula I having the following structure:

or salts thereof, wherein:

-   -   R¹ and R² are either the same or different and are independently        hydrogen (H) or an optionally substituted C₁-C₆ alkyl, C₂-C₆        alkenyl, or C₂-C₆ alkynyl, or R¹ and R² may join to form an        optionally substituted heterocyclic ring;    -   R³ is either absent or, if present, is hydrogen (H) or a C₁-C₆        alkyl to provide a quaternary amine;    -   R⁴ and R⁵ are either the same or different and are independently        an optionally substituted C₁₀-C₂₄ alkyl, C₁₀-C₂₄ alkenyl,        C₁₀-C₂₄ alkynyl, or C₁₀-C₂₄ acyl, wherein at least one of R⁴ and        R⁵ comprises at least one optionally substituted cyclic alkyl        group;    -   X and Y are either the same or different and are independently        O, S, N(R⁶), C(O), C(O)O, OC(O), C(O)N(R⁶), N(R⁶)C(O),        OC(O)N(R⁶), N(R⁶)C(O)O, C(O)S, C(S)O, S(O), S(O)(O), C(S), or an        optionally substituted heterocyclic ring, wherein R⁶ is        hydrogen (H) or an optionally substituted C₁-C₁₀ alkyl, C₂-C₁₀        alkenyl, or C₂-C₁₀ alkynyl; and    -   Z is either absent or, if present, is an optionally substituted        C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl.

In one embodiment, R¹ and R² are independently selected from the groupconsisting of a methyl group and an ethyl group, i.e., R¹ and R² areboth methyl groups, R¹ and R² are both ethyl groups, or R¹ and R² are acombination of one methyl group and one ethyl group. In anotherembodiment, R¹ and R² are joined to form an optionally substitutedheterocyclic ring having from 2 to 5 carbon atoms (e.g., 2, 3, 4, or 5carbon atoms, or from 2-5, 2-4, 2-3, 3-5, 3-4, or 4-5 carbon atoms) andfrom 1 to 3 heteroatoms (e.g., 1, 2, or 3 heteroatoms, or from 1-3, 1-2,or 2-3 heteroatoms) selected from the group consisting of nitrogen (N),oxygen (O), sulfur (S), and combinations thereof. In certain instances,R¹ and R² are joined to form an optionally substituted diazole (e.g., anoptionally substituted imidazole) or an optionally substituted triazole.

In yet another embodiment, X and Y are either the same or different andare independently O, C(O)O, C(O)N(R⁶), N(R⁶)C(O)O, or C(O)S. In certaininstances, R⁶ is hydrogen (H), an optionally substituted methyl group,an optionally substituted ethyl group, or an optionally substitutedC₃-C₁₀ alkyl, alkenyl, or alkynyl group (e.g., an optionally substitutedC₃, C₄, C₅, C₆, C₇, C₈, C₉, or C₁₀ alkyl, alkenyl, or alkynyl group). Ina further embodiment, X and Y are independently an optionallysubstituted heterocyclic ring having from 2 to 5 carbon atoms (e.g., 2,3, 4, or 5 carbon atoms, or from 2-5, 2-4, 2-3, 3-5, 3-4, or 4-5 carbonatoms) and from 1 to 3 heteroatoms (e.g., 1, 2, or 3 heteroatoms, orfrom 1-3, 1-2, or 2-3 heteroatoms) selected from the group consisting ofnitrogen (N), oxygen (O), sulfur (S), and combinations thereof. Incertain instances, X and Y are independently an optionally substituteddiazole (e.g., an optionally substituted imidazole) or an optionallysubstituted triazole. In preferred embodiments, X and Y are both O. Inother embodiments, Z is (CH₂)_(n) and n is 0, 1, 2, 3, 4, 5, or 6. Inparticular embodiments, n is 1, 2, 3, or 4 (e.g., n is 1-4, 1-3, 1-2,2-4, 2-3, or 3-4).

In certain embodiments, at least one of R⁴ and R⁵ comprises at leastone, two, three, or more optionally substituted cyclic alkyl groups. Inparticular embodiments, both R⁴ and R⁵ independently comprise at leastone, two, three, or more optionally substituted cyclic alkyl groups. Insome instances, both R⁴ and R⁵ comprise the same number of (e.g., 1, 2,3, 4, 5, 6, or more) optionally substituted cyclic alkyl groups. Inother instances, R⁴ and R⁵ comprise a different number of optionallysubstituted cyclic alkyl groups. In one embodiment, each of theoptionally substituted cyclic alkyl groups in R⁴ and/or R⁵ comprises anindependently selected optionally substituted saturated cyclic alkylgroup or an optionally substituted unsaturated cyclic alkyl group. Incertain instances, at least one, two, three, or more of the optionallysubstituted cyclic alkyl groups present in one or both of R⁴ and R⁵independently comprises an optionally substituted C₃₋₈ cycloalkyl groupsuch as, e.g., a cyclopropyl group, an optionally substituted C₃₋₈cycloalkenyl group, and combinations thereof. In some embodiments, oneof R⁴ or R⁵ comprises at least one, two, three, or more optionallysubstituted cyclic alkyl groups and the other side-chain comprises anoptionally substituted C₁₀-C₂₄ alkyl, C₁₀-C₂₄ alkenyl, C₁₀-C₂₄ alkynyl,or C₁₀-C₂₄ acyl group (e.g., a side-chain comprising at least one, two,or three sites of unsaturation). In some instances, R⁴ and R⁵ bothcomprise the same type or types of optionally substituted cyclic alkylgroups. In other instances, R⁴ and R⁵ comprise different types ofoptionally substituted cyclic alkyl groups.

In particular embodiments, R⁴ and R⁵ are both C₁₂-C₂₀ alkyl groups(e.g., C₁₈ alkyl groups) having at least one, two, three, or moreoptionally substituted cyclic alkyl groups. In preferred embodiments, R⁴and R⁵ are both C₁₂-C₂₀ (e.g., C₁₈) alkyl groups having the same numberof (e.g., at least one, two, three, or more) optionally substitutedcyclic alkyl groups. In certain embodiments, the at least one, two,three, or more optionally substituted cyclic alkyl groups present inboth R⁴ and R⁵ independently comprises an optionally substitutedsaturated cyclic alkyl group (e.g., a C₃₋₈ cycloalkyl group such as acyclopropyl group) or an optionally substituted unsaturated cyclic alkylgroup (e.g., a C₃₋₈ cycloalkenyl group).

In some embodiments, at least one, two, three, or more optionallysubstituted cyclic alkyl groups are present on each of R⁴ and/or R⁵ incombination with at least one, two, three, or more sites of unsaturationand/or branched alkyl and/or acyl groups. For example, R⁴ may compriseone, two, or three C₃₋₈ cycloalkyl groups such as cyclopropyl groups andone, two, or three sites of unsaturation, while R⁵ may comprise the sameor different number and type of substituents.

In particular embodiments, the cationic lipid of Formula I has one ofthe following structures:

In another aspect, the present invention provides a cationic lipid ofFormula II having the following structure:

or salts thereof, wherein:

-   -   R¹ and R² are either the same or different and are independently        an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆        alkynyl, or R¹ and R² may join to form an optionally substituted        heterocyclic ring;    -   R³ is either absent or, if present, is hydrogen (H) or a C₁-C₆        alkyl to provide a quaternary amine;    -   R⁴ and R⁵ are either the same or different and are independently        an optionally substituted C₁₀-C₂₄ alkyl, C₁₀-C₂₄ alkenyl,        C₁₀-C₂₄ alkynyl, or C₁₀-C₂₄ acyl, wherein at least one of R⁴ and        R⁵ comprises at least one optionally substituted cyclic alkyl        group;    -   m, n, and p are either the same or different and are        independently either 0, 1, or 2, with the proviso that m, n, and        p are not simultaneously 0;    -   X and Y are either the same or different and are independently        O, S, N(R⁶), C(O), C(O)O, OC(O), C(O)N(R), N(R⁶)C(O),        OC(O)N(R⁶), N(R⁶)C(O)O, C(O)S, C(S)O, S(O), S(O)(O), or C(S),        wherein R⁶ is hydrogen (H) or an optionally substituted C₁-C₁₀        alkyl, C₂-C₁₀ alkenyl, or C₂-C₁₀ alkynyl; and    -   Z is either absent or, if present, is an optionally substituted        C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl.

In one embodiment, R¹ and R² are independently selected from the groupconsisting of a methyl group and an ethyl group, i.e., R¹ and R² areboth methyl groups, R¹ and R² are both ethyl groups, or R¹ and R² are acombination of one methyl group and one ethyl group. In anotherembodiment, R¹ and R² are joined to form an optionally substitutedheterocyclic ring having from 2 to 5 carbon atoms (e.g., 2, 3, 4, or 5carbon atoms, or from 2-5, 2-4, 2-3, 3-5, 3-4, or 4-5 carbon atoms) andfrom 1 to 3 heteroatoms (e.g., 1, 2, or 3 heteroatoms, or from 1-3, 1-2,or 2-3 heteroatoms) selected from the group consisting of nitrogen (N),oxygen (O), sulfur (S), and combinations thereof. In certain instances,R¹ and R² are joined to form an optionally substituted diazole (e.g., anoptionally substituted imidazole) or an optionally substituted triazole.

In yet another embodiment, X and Y are either the same or different andare independently O, C(O)O, C(O)N(R⁶), N(R⁶)C(O)O, or C(O)S. In certaininstances, R⁶ is hydrogen (H), an optionally substituted methyl group,an optionally substituted ethyl group, or an optionally substitutedC₃-C₁₀ alkyl, alkenyl, or alkynyl group (e.g., an optionally substitutedC₃, C₄, C₅, C₆, C₇, C₈, C₉, or C₁₀ alkyl, alkenyl, or alkynyl group). Inpreferred embodiments, X and Y are both O. In other embodiments, Z is(CH₂)_(q) and q is 0, 1, 2, 3, 4, 5, or 6. In particular embodiments, qis 1, 2, 3, or 4 (e.g., q is 1-4, 1-3, 1-2, 2-4, 2-3, or 3-4).

In certain embodiments, at least one of R⁴ and R⁵ comprises at leastone, two, three, or more optionally substituted cyclic alkyl groups. Inparticular embodiments, both R⁴ and R⁵ independently comprise at leastone, two, three, or more optionally substituted cyclic alkyl groups. Insome instances, both R⁴ and R⁵ comprise the same number of (e.g., 1, 2,3, 4, 5, 6, or more) optionally substituted cyclic alkyl groups. Inother instances, R⁴ and R⁵ comprise a different number of optionallysubstituted cyclic alkyl groups. In one embodiment, each of theoptionally substituted cyclic alkyl groups in R⁴ and/or R⁵ comprises anindependently selected optionally substituted saturated cyclic alkylgroup or an optionally substituted unsaturated cyclic alkyl group. Incertain instances, at least one, two, three, or more of the optionallysubstituted cyclic alkyl groups present in one or both of R⁴ and R⁵independently comprises an optionally substituted C₃₋₈ cycloalkyl groupsuch as, e.g., a cyclopropyl group, an optionally substituted C₃₋₈cycloalkenyl group, and combinations thereof. In some embodiments, oneof R⁴ or R⁵ comprises at least one, two, three, or more optionallysubstituted cyclic alkyl groups and the other side-chain comprises anoptionally substituted C₁₀-C₂₄ alkyl, C₁₀-C₂₄ alkenyl, C₁₀-C₂₄ alkynyl,or C₁₀-C₂₄ acyl group (e.g., a side-chain comprising at least one, two,or three sites of unsaturation). In some instances, R⁴ and R⁵ bothcomprise the same type or types of optionally substituted cyclic alkylgroups. In other instances, R⁴ and R⁵ comprise different types ofoptionally substituted cyclic alkyl groups.

In particular embodiments, R⁴ and R⁵ are both C₁₂-C₂₀ alkyl groups(e.g., C₁₈ alkyl groups) having at least one, two, three, or moreoptionally substituted cyclic alkyl groups. In preferred embodiments, R⁴and R⁵ are both C₁₂-C₂₀ (e.g., C₁₈) alkyl groups having the same numberof (e.g., at least one, two, three, or more) optionally substitutedcyclic alkyl groups. In certain embodiments, the at least one, two,three, or more optionally substituted cyclic alkyl groups present inboth R⁴ and R⁵ independently comprises an optionally substitutedsaturated cyclic alkyl group (e.g., a C₃₋₈ cycloalkyl group such as acyclopropyl group) or an optionally substituted unsaturated cyclic alkylgroup (e.g., a C₃₋₈ cycloalkenyl group).

In some embodiments, at least one, two, three, or more optionallysubstituted cyclic alkyl groups are present on each of R⁴ and/or R⁵ incombination with at least one, two, three, or more sites of unsaturationand/or branched alkyl and/or acyl groups. For example, R⁴ may compriseone, two, or three C₃₋₈ cycloalkyl groups such as cyclopropyl groups andone, two, or three sites of unsaturation, while R⁵ may comprise the sameor different number and type of substituents.

In particular embodiments, the cationic lipid of Formula II has one ofthe following structures:

In yet another aspect, the present invention provides a cationic lipidof Formula III having the following structure:

or salts thereof, wherein:

-   -   R¹ and R² are either the same or different and are independently        hydrogen (H) or an optionally substituted C₁-C₆ alkyl, C₂-C₆        alkenyl, or C₂-C₆ alkynyl, or R¹ and R² may join to form an        optionally substituted heterocyclic ring;    -   R³ is either absent or, if present, is hydrogen (H) or a C₁-C₆        alkyl to provide a quaternary amine;    -   R⁴ and R⁵ are either the same or different and are independently        an optionally substituted C₁₀-C₂₄ alkyl, C₁₀-C₂₄ alkenyl,        C₁₀-C₂₄ alkynyl, or C₁₀-C₂₄ acyl, wherein at least one of R⁴ and        R⁵ comprises at least one optionally substituted cyclic alkyl        group;    -   X is O, S, N(R⁶), C(O), C(O)O, OC(O), C(O)N(R⁶), N(R⁶)C(O),        OC(O)N(R⁶), N(R⁶)C(O)O, C(O)S, C(S)O, S(O), S(O)(O), C(S), or an        optionally substituted heterocyclic ring, wherein R⁶ is        hydrogen (H) or an optionally substituted C₁-C₁₀ alkyl, C₂-C₁₀        alkenyl, or C₂-C₁₀ alkynyl; and    -   Y is either absent or, if present, is an optionally substituted        C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl.

In one embodiment, R¹ and R² are independently selected from the groupconsisting of a methyl group and an ethyl group, i.e., R¹ and R² areboth methyl groups, R¹ and R² are both ethyl groups, or R¹ and R² are acombination of one methyl group and one ethyl group.

In another embodiment, R¹ and R² are joined to form an optionallysubstituted heterocyclic ring having from 2 to 5 carbon atoms (e.g., 2,3, 4, or 5 carbon atoms, or from 2-5, 2-4, 2-3, 3-5, 3-4, or 4-5 carbonatoms) and from 1 to 3 heteroatoms (e.g., 1, 2, or 3 heteroatoms, orfrom 1-3, 1-2, or 2-3 heteroatoms) selected from the group consisting ofnitrogen (N), oxygen (O), sulfur (S), and combinations thereof. Incertain instances, R¹ and R² are joined to form an optionallysubstituted diazole (e.g., an optionally substituted imidazole) or anoptionally substituted triazole.

In yet another embodiment, X is O, C(O)O, C(O)N(R⁶), N(R⁶)C(O)O, orC(O)S. In certain instances, R⁶ is hydrogen (H), an optionallysubstituted methyl group, an optionally substituted ethyl group, or anoptionally substituted C₃-C₁₀ alkyl, alkenyl, or alkynyl group (e.g., anoptionally substituted C₃, C₄, C₅, C₆, C₇, C₈, C₉, or C₁₀ alkyl,alkenyl, or alkynyl group). In a further embodiment, X is an optionallysubstituted heterocyclic ring having from 2 to 5 carbon atoms (e.g., 2,3, 4, or 5 carbon atoms, or from 2-5, 2-4, 2-3, 3-5, 3-4, or 4-5 carbonatoms) and from 1 to 3 heteroatoms (e.g., 1, 2, or 3 heteroatoms, orfrom 1-3, 1-2, or 2-3 heteroatoms) selected from the group consisting ofnitrogen (N), oxygen (O), sulfur (S), and combinations thereof. Incertain instances, X is an optionally substituted diazole (e.g., anoptionally substituted imidazole) or an optionally substituted triazole.In preferred embodiments, X is C(O)O. In other embodiments, Y is(CH₂)_(n) and n is 0, 1, 2, 3, 4, 5, or 6. In particular embodiments, nis 1, 2, 3, or 4 (e.g., n is 1-4, 1-3, 1-2, 2-4, 2-3, or 3-4).

In certain embodiments, at least one of R⁴ and R⁵ comprises at leastone, two, three, or more optionally substituted cyclic alkyl groups. Inparticular embodiments, both R⁴ and R⁵ independently comprise at leastone, two, three, or more optionally substituted cyclic alkyl groups. Insome instances, both R⁴ and R⁵ comprise the same number of (e.g., 1, 2,3, 4, 5, 6, or more) optionally substituted cyclic alkyl groups. Inother instances, R⁴ and R⁵ comprise a different number of optionallysubstituted cyclic alkyl groups. In one embodiment, each of theoptionally substituted cyclic alkyl groups in R⁴ and/or R⁵ comprises anindependently selected optionally substituted saturated cyclic alkylgroup or an optionally substituted unsaturated cyclic alkyl group. Incertain instances, at least one, two, three, or more of the optionallysubstituted cyclic alkyl groups present in one or both of R⁴ and R⁵independently comprises an optionally substituted C₃₋₈ cycloalkyl groupsuch as, e.g., a cyclopropyl group, an optionally substituted C₃₋₈cycloalkenyl group, and combinations thereof. In some embodiments, oneof R⁴ or R⁵ comprises at least one, two, three, or more optionallysubstituted cyclic alkyl groups and the other side-chain comprises anoptionally substituted C₁₀-C₂₄ alkyl, C₁₀-C₂₄ alkenyl, C₁₀-C₂₄ alkynyl,or C₁₀-C₂₄ acyl group (e.g., a side-chain comprising at least one, two,or three sites of unsaturation). In some instances, R⁴ and R⁵ bothcomprise the same type or types of optionally substituted cyclic alkylgroups. In other instances, R⁴ and R⁵ comprise different types ofoptionally substituted cyclic alkyl groups.

In particular embodiments, R⁴ and R⁵ are both C₁₂-C₂₀ alkyl groups(e.g., C₁₈ alkyl groups) having at least one, two, three, or moreoptionally substituted cyclic alkyl groups. In preferred embodiments, R⁴and R⁵ are both C₁₂-C₂₀ (e.g., C₁₈) alkyl groups having the same numberof (e.g., at least one, two, three, or more) optionally substitutedcyclic alkyl groups. In certain embodiments, the at least one, two,three, or more optionally substituted cyclic alkyl groups present inboth R⁴ and R⁵ independently comprises an optionally substitutedsaturated cyclic alkyl group (e.g., a C₃₋₈ cycloalkyl group such as acyclopropyl group) or an optionally substituted unsaturated cyclic alkylgroup (e.g., a C₃₋₈ cycloalkenyl group).

In some embodiments, at least one, two, three, or more optionallysubstituted cyclic alkyl groups are present on each of R⁴ and/or R⁵ incombination with at least one, two, three, or more sites of unsaturationand/or branched alkyl and/or acyl groups. For example, R⁴ may compriseone, two, or three C₃₋₈ cycloalkyl groups such as cyclopropyl groups andone, two, or three sites of unsaturation, while R⁵ may comprise the sameor different number and type of substituents.

In particular embodiments, the cationic lipid of Formula III has one ofthe following structures:

In a further aspect, the present invention provides a lipid particlecomprising one or more of the above cationic lipids of Formulas I-III orsalts thereof. In certain embodiments, the lipid particle furthercomprises one or more non-cationic lipids such as neutral lipids. Incertain other embodiments, the lipid particle further comprises one ormore conjugated lipids capable of reducing or inhibiting particleaggregation. In additional embodiments, the lipid particle furthercomprises one or more active agents or therapeutic agents.

In certain embodiments, the non-cationic lipid component of the lipidparticle may comprise a phospholipid, cholesterol (or cholesterolderivative), or a mixture thereof. In one particular embodiment, thephospholipid comprises dipalmitoylphosphatidylcholine (DPPC),distearoylphosphatidylcholine (DSPC), or a mixture thereof. In someembodiments, the conjugated lipid component of the lipid particlecomprises a polyethyleneglycol (PEG)-lipid conjugate. In certaininstances, the PEG-lipid conjugate comprises a PEG-diacylglycerol(PEG-DAG) conjugate, a PEG-dialkyloxypropyl (PEG-DAA) conjugate, or amixture thereof. In other embodiments, the lipid conjugate comprises apolyoxazoline (POZ)-lipid conjugate such as a POZ-DAA conjugate.

In some embodiments, the active agent or therapeutic agent comprises anucleic acid. In certain instances, the nucleic acid comprises aninterfering RNA molecule which includes any double-stranded RNA capableof mediating RNAi, such as, e.g., an siRNA, Dicer-substrate dsRNA,shRNA, aiRNA, pre-miRNA, or mixtures thereof. In certain otherinstances, the nucleic acid comprises single-stranded or double-strandedDNA, RNA, or a DNA/RNA hybrid such as, e.g., an antisenseoligonucleotide, a ribozyme, a plasmid, an immunostimulatoryoligonucleotide, or mixtures thereof.

In other embodiments, the active agent or therapeutic agent is fullyencapsulated within the lipid portion of the lipid particle such thatthe active agent or therapeutic agent in the lipid particle is resistantin aqueous solution to enzymatic degradation, e.g., by a nuclease orprotease. In further embodiments, the lipid particle is substantiallynon-toxic to mammals such as humans.

In preferred embodiments, the present invention provides serum-stablenucleic acid-lipid particles (SNALP) comprising: (a) one or more nucleicacids such as interfering RNA molecules; (b) one or more cationic lipidsof Formulas I-III or salts thereof; (c) one or more non-cationic lipids;and (d) one or more conjugated lipids that inhibit aggregation ofparticles.

In some embodiments, the present invention provides nucleic acid-lipidparticles (e.g., SNALP) comprising: (a) one or more nucleic acids; (b)one or more cationic lipids of Formulas I-III or salts thereofcomprising from about 50 mol % to about 85 mol % of the total lipidpresent in the particle; (c) one or more non-cationic lipids comprisingfrom about 13 mol % to about 49.5 mol % of the total lipid present inthe particle; and (d) one or more conjugated lipids that inhibitaggregation of particles comprising from about 0.5 mol % to about 2 mol% of the total lipid present in the particle.

In one aspect of this embodiment, the nucleic acid-lipid particlecomprises: (a) one or more nucleic acids; (b) one or more cationiclipids of Formulas I-III or a salt thereof comprising from about 50 mol% to about 65 mol % of the total lipid present in the particle; (c) oneor more non-cationic lipids comprising a mixture of one or morephospholipids and cholesterol or a derivative thereof, wherein the oneor more phospholipids comprises from about 4 mol % to about 10 mol % ofthe total lipid present in the particle and the cholesterol orderivative thereof comprises from about 30 mol % to about 40 mol % ofthe total lipid present in the particle; and (d) one or more PEG-lipidconjugates comprising from about 0.5 mol % to about 2 mol % of the totallipid present in the particle. This embodiment of nucleic acid-lipidparticle is generally referred to herein as the “1:57” formulation.

In certain instances, the 1:57 formulation comprises: (a) one or morenucleic acids; (b) one or more cationic lipids of Formulas I-III or asalt thereof comprising from about 52 mol % to about 62 mol % of thetotal lipid present in the particle; (c) a mixture of one or morephospholipids and cholesterol or a derivative thereof comprising fromabout 36 mol % to about 47 mol % of the total lipid present in theparticle; and (d) one or more PEG-lipid conjugates comprising from about1 mol % to about 2 mol % of the total lipid present in the particle. Inone particular embodiment, the 1:57 formulation is a four-componentsystem comprising about 1.4 mol % PEG-lipid conjugate (e.g.,PEG2000-C-DMA), about 57.1 mol % cationic lipid of Formulas I-III or asalt thereof, about 7.1 mol % DPPC (or DSPC), and about 34.3 mol %cholesterol (or derivative thereof).

In another aspect of this embodiment, the nucleic acid-lipid particlecomprises: (a) one or more nucleic acids; (b) one or more cationiclipids of Formulas I-III or a salt thereof comprising from about 56.5mol % to about 66.5 mol % of the total lipid present in the particle;(c) cholesterol and/or one or more derivatives thereof comprising fromabout 31.5 mol % to about 42.5 mol % of the total lipid present in theparticle; and (d) one or more PEG-lipid conjugates comprising from about1 mol % to about 2 mol % of the total lipid present in the particle.This embodiment of nucleic acid-lipid particle is generally referred toherein as the “1:62” formulation. In one particular embodiment, the 1:62formulation is a three-component system which is phospholipid-free andcomprises about 1.5 mol % PEG-lipid conjugate (e.g., PEG2000-C-DMA),about 61.5 mol % cationic lipid of Formulas I-III or a salt thereof, andabout 36.9 mol % cholesterol (or derivative thereof).

Additional embodiments related to the 1:57 and 1:62 formulations aredescribed in PCT Publication No. WO 09/127060 and U.S. Publication No.20110071208, the disclosures of which are herein incorporated byreference in their entirety for all purposes.

In other embodiments, the present invention provides nucleic acid-lipidparticles (e.g., SNALP) comprising: (a) one or more nucleic acids; (b)one or more cationic lipids of Formulas I-III or salts thereofcomprising from about 2 mol % to about 50 mol % of the total lipidpresent in the particle; (c) one or more non-cationic lipids comprisingfrom about 5 mol % to about 90 mol % of the total lipid present in theparticle; and (d) one or more conjugated lipids that inhibit aggregationof particles comprising from about 0.5 mol % to about 20 mol % of thetotal lipid present in the particle.

In one aspect of this embodiment, the nucleic acid-lipid particlecomprises: (a) one or more nucleic acids; (b) one or more cationiclipids of Formulas I-III or a salt thereof comprising from about 30 mol% to about 50 mol % of the total lipid present in the particle; (c) amixture of one or more phospholipids and cholesterol or a derivativethereof comprising from about 47 mol % to about 69 mol % of the totallipid present in the particle; and (d) one or more PEG-lipid conjugatescomprising from about 1 mol % to about 3 mol % of the total lipidpresent in the particle. This embodiment of nucleic acid-lipid particleis generally referred to herein as the “2:40” formulation. In oneparticular embodiment, the 2:40 formulation is a four-component systemwhich comprises about 2 mol % PEG-lipid conjugate (e.g., PEG2000-C-DMA),about 40 mol % cationic lipid of Formulas I-III or a salt thereof, about10 mol % DPPC (or DSPC), and about 48 mol % cholesterol (or derivativethereof).

In further embodiments, the present invention provides nucleicacid-lipid particles (e.g., SNALP) comprising: (a) one or more nucleicacids; (b) one or more cationic lipids of Formulas I-III or saltsthereof comprising from about 50 mol % to about 65 mol % of the totallipid present in the particle; (c) one or more non-cationic lipidscomprising from about 25 mol % to about 45 mol % of the total lipidpresent in the particle; and (d) one or more conjugated lipids thatinhibit aggregation of particles comprising from about 5 mol % to about10 mol % of the total lipid present in the particle.

In one aspect of this embodiment, the nucleic acid-lipid particlecomprises: (a) one or more nucleic acids; (b) one or more cationiclipids of Formulas I-III or a salt thereof comprising from about 50 mol% to about 60 mol % of the total lipid present in the particle; (c) amixture of one or more phospholipids and cholesterol or a derivativethereof comprising from about 35 mol % to about 45 mol % of the totallipid present in the particle; and (d) one or more PEG-lipid conjugatescomprising from about 5 mol % to about 10 mol % of the total lipidpresent in the particle. This embodiment of nucleic acid-lipid particleis generally referred to herein as the “7:54” formulation. In certaininstances, the non-cationic lipid mixture in the 7:54 formulationcomprises: (i) a phospholipid of from about 5 mol % to about 10 mol % ofthe total lipid present in the particle; and (ii) cholesterol or aderivative thereof of from about 25 mol % to about 35 mol % of the totallipid present in the particle. In one particular embodiment, the 7:54formulation is a four-component system which comprises about 7 mol %PEG-lipid conjugate (e.g., PEG750-C-DMA), about 54 mol % cationic lipidof Formulas I-III or a salt thereof, about 7 mol % DPPC (or DSPC), andabout 32 mol % cholesterol (or derivative thereof).

In another aspect of this embodiment, the nucleic acid-lipid particlecomprises: (a) one or more nucleic acids; (b) one or more cationiclipids of Formulas I-III or a salt thereof comprising from about 55 mol% to about 65 mol % of the total lipid present in the particle; (c)cholesterol and/or one or more derivatives thereof comprising from about30 mol % to about 40 mol % of the total lipid present in the particle;and (d) one or more PEG-lipid conjugates comprising from about 5 mol %to about 10 mol % of the total lipid present in the particle. Thisembodiment of nucleic acid-lipid particle is generally referred toherein as the “7:58” formulation. In one particular embodiment, the 7:58formulation is a three-component system which is phospholipid-free andcomprises about 7 mol % PEG-lipid conjugate (e.g., PEG750-C-DMA), about58 mol % cationic lipid of Formulas I-III or a salt thereof, and about35 mol % cholesterol (or derivative thereof).

Additional embodiments related to the 7:54 and 7:58 formulations aredescribed in U.S. Patent Publication No. 20110076335, the disclosure ofwhich is herein incorporated by reference in its entirety for allpurposes.

The present invention also provides pharmaceutical compositionscomprising a lipid particle such as a nucleic acid-lipid particle (e.g.,SNALP) and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides methods forintroducing one or more therapeutic agents such as nucleic acids into acell, the method comprising contacting the cell with a lipid particledescribed herein (e.g., SNALP). In one embodiment, the cell is in amammal and the mammal is a human.

In yet another aspect, the present invention provides methods for the invivo delivery of one or more therapeutic agents such as nucleic acids,the method comprising administering to a mammal a lipid particledescribed herein (e.g., SNALP). In certain embodiments, the lipidparticles (e.g., SNALP) are administered by one of the following routesof administration: oral, intranasal, intravenous, intraperitoneal,intramuscular, intraarticular, intralesional, intratracheal,subcutaneous, and intradermal. In particular embodiments, the lipidparticles (e.g., SNALP) are administered systemically, e.g., via enteralor parenteral routes of administration. In preferred embodiments, themammal is a human.

In a further aspect, the present invention provides methods for treatinga disease or disorder in a mammal in need thereof, the method comprisingadministering to the mammal a therapeutically effective amount of alipid particle (e.g., SNALP) comprising one or more therapeutic agentssuch as nucleic acids. Non-limiting examples of diseases or disordersinclude a viral infection, a liver disease or disorder, and cancer.Preferably, the mammal is a human.

In certain embodiments, the present invention provides methods fortreating a liver disease or disorder by administering a nucleic acidsuch as an interfering RNA (e.g., siRNA) in nucleic acid-lipid particles(e.g., SNALP), alone or in combination with a lipid-lowering agent.Examples of lipid diseases and disorders include, but are not limitedto, dyslipidemia (e.g., hyperlipidemias such as elevated triglyceridelevels (hypertriglyceridemia) and/or elevated cholesterol levels(hypercholesterolemia)), atherosclerosis, coronary heart disease,coronary artery disease, atherosclerotic cardiovascular disease (CVD),fatty liver disease (hepatic steatosis), abnormal lipid metabolism,abnormal cholesterol metabolism, diabetes (including Type 2 diabetes),obesity, cardiovascular disease, and other disorders relating toabnormal metabolism. Non-limiting examples of lipid-lowering agentsinclude statins, fibrates, ezetimibe, thiazolidinediones, niacin,beta-blockers, nitroglycerin, calcium antagonists, and fish oil.

In one particular embodiment, the present invention provides a methodfor lowering or reducing cholesterol levels in a mammal (e.g., human) inneed thereof (e.g., a mammal with elevated blood cholesterol levels),the method comprising administering to the mammal a therapeuticallyeffective amount of a nucleic acid-lipid particle (e.g., a SNALPformulation) described herein comprising one or more interfering RNAs(e.g., siRNAs) that target one or more genes associated with metabolicdiseases and disorders. In another particular embodiment, the presentinvention provides a method for lowering or reducing triglyceride levelsin a mammal (e.g., human) in need thereof (e.g., a mammal with elevatedblood triglyceride levels), the method comprising administering to themammal a therapeutically effective amount of a nucleic acid-lipidparticle (e.g., a SNALP formulation) described herein comprising one ormore interfering RNAs (e.g., siRNAs) that target one or more genesassociated with metabolic diseases and disorders. These methods can becarried out in vitro using standard tissue culture techniques or in vivoby administering the interfering RNA (e.g., siRNA) using any means knownin the art. In preferred embodiments, the interfering RNA (e.g., siRNA)is delivered to a liver cell (e.g., hepatocyte) in a mammal such as ahuman.

Additional embodiments related to treating a liver disease or disorderusing a lipid particle are described in, e.g., PCT Publication No. WO2010/083615, and U.S. Patent Publication No. 20060134189, thedisclosures of which are herein incorporated by reference in theirentirety for all purposes.

In other embodiments, the present invention provides methods fortreating a cell proliferative disorder such as cancer by administering anucleic acid such as an interfering RNA (e.g., siRNA) in nucleicacid-lipid particles (e.g., SNALP), alone or in combination with achemotherapy drug. The methods can be carried out in vitro usingstandard tissue culture techniques or in vivo by administering theinterfering RNA (e.g., siRNA) using any means known in the art. Inpreferred embodiments, the interfering RNA (e.g., siRNA) is delivered toa cancer cell in a mammal such as a human, alone or in combination witha chemotherapy drug. The nucleic acid-lipid particles and/orchemotherapy drugs may also be co-administered with conventionalhormonal, immunotherapeutic, and/or radiotherapeutic agents.

Additional embodiments related to treating a cell proliferative disorderusing a lipid particle are described in, e.g., PCT Publication No. WO09/082817, U.S. Patent Publication No. 20090149403, PCT Publication No.WO 09/129319, and PCT Publication No. WO 2011/038160, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes.

In further embodiments, the present invention provides methods forpreventing or treating a viral infection such as an arenavirus (e.g.,Lassa virus) or filovirus (e.g., Ebola virus, Marburg virus, etc.)infection which causes hemorrhagic fever or a hepatitis (e.g., HepatitisC virus) infection which causes acute or chronic hepatitis byadministering a nucleic acid such as an interfering RNA (e.g., siRNA) innucleic acid-lipid particles (e.g., SNALP), alone or in combination withthe administration of conventional agents used to treat or amelioratethe viral condition or any of the symptoms associated therewith. Themethods can be carried out in vitro using standard tissue culturetechniques or in vivo by administering the interfering RNA using anymeans known in the art. In certain preferred embodiments, theinterfering RNA (e.g., siRNA) is delivered to cells, tissues, or organsof a mammal such as a human that are infected and/or susceptible ofbeing infected with the hemorrhagic fever virus, such as, e.g., cells ofthe reticuloendothelial system (e.g., monocytes, macrophages, etc.),endothelial cells, liver cells (e.g., hepatocytes), fibroblast cells,and/or platelet cells. In certain other preferred embodiments, theinterfering RNA (e.g., siRNA) is delivered to cells, tissues, or organsof a mammal such as a human that are infected and/or susceptible ofbeing infected with the hepatitis virus, such as, e.g., cells of theliver (e.g., hepatocytes).

Additional embodiments related to preventing or treating a viralinfection using a lipid particle are described in, e.g., U.S. PatentPublication No. 20070218122, U.S. Patent Publication No. 20070135370,PCT Publication No. WO 2011/011447, PCT Publication No. WO 2010/105372,and U.S. patent application Ser. No. 13/077,856, filed Mar. 31, 2011,the disclosures of which are herein incorporated by reference in theirentirety for all purposes.

The lipid particles of the invention (e.g., SNALP) comprising one ormore cationic lipids of Formulas I-III or salts thereof are particularlyadvantageous and suitable for use in the administration of nucleic acidssuch as interfering RNA to a subject (e.g., a mammal such as a human)because they are stable in circulation, of a size required forpharmacodynamic behavior resulting in access to extravascular sites, andare capable of reaching target cell populations.

Other objects, features, and advantages of the present invention will beapparent to one of skill in the art from the following detaileddescription and figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the apparent pK_(a) values of exemplary SNALP formulationscontaining cationic lipids of Formula I.

FIG. 2 shows the apparent pK_(a) values of exemplary SNALP formulationscontaining cationic lipids of Formula III.

FIG. 3 shows a comparison of the liver ApoB mRNA knockdown activity ofexemplary SNALP formulations containing cationic lipids of Formula I.

FIG. 4 shows a comparison of the liver ApoB mRNA knockdown activity ofexemplary SNALP formulations containing cationic lipids of FormulasII-III.

FIG. 5 shows a comparison of the liver ApoB mRNA knockdown activity ofexemplary SNALP formulations containing cationic lipids of Formula III.

FIG. 6 shows the KD50 calculation and values obtained for each of theexemplary SNALP formulations containing cationic lipids of Formula III.

FIG. 7 shows a comparison of the liver ApoB mRNA knockdown activity ofadditional exemplary SNALP formulations containing cationic lipids ofFormulas II-III.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present invention is based, in part, upon the discovery of novelcationic (amino) lipids that provide advantages when used in lipidparticles for the in vivo delivery of an active or therapeutic agentsuch as a nucleic acid into a cell of a mammal. In particular, thepresent invention provides nucleic acid-lipid particle compositionscomprising one or more of the novel cationic lipids described hereinthat provide increased activity of the nucleic acid (e.g., interferingRNA) and improved tolerability of the compositions in vivo, resulting ina significant increase in the therapeutic index as compared to nucleicacid-lipid particle compositions previously described.

In particular embodiments, the present invention provides novel cationiclipids that enable the formulation of improved compositions for the invitro and in vivo delivery of interfering RNA such as siRNA. It is shownherein that these improved lipid particle compositions are effective indown-regulating (e.g., silencing) the protein levels and/or mRNA levelsof target genes. Furthermore, it is shown herein that the activity ofthese improved lipid particle compositions is dependent on the presenceof the novel cationic lipids of the invention.

The lipid particles and compositions of the present invention may beused for a variety of purposes, including the delivery of encapsulatedor associated (e.g., complexed) therapeutic agents such as nucleic acidsto cells, both in vitro and in vivo. Accordingly, the present inventionfurther provides methods of treating diseases or disorders in a subjectin need thereof by contacting the subject with a lipid particle thatencapsulates or is associated with a suitable therapeutic agent, whereinthe lipid particle comprises one or more of the novel cationic lipidsdescribed herein.

As described herein, the lipid particles of the present invention areparticularly useful for the delivery of nucleic acids, including, e.g.,interfering RNA molecules such as siRNA. Therefore, the lipid particlesand compositions of the present invention may be used to decrease theexpression of target genes and proteins both in vitro and in vivo bycontacting cells with a lipid particle comprising one or more novelcationic lipids described herein, wherein the lipid particleencapsulates or is associated with a nucleic acid that reduces targetgene expression (e.g., an siRNA). Alternatively, the lipid particles andcompositions of the present invention may be used to increase theexpression of a desired protein both in vitro and in vivo by contactingcells with a lipid particle comprising one or more novel cationic lipidsdescribed herein, wherein the lipid particle encapsulates or isassociated with a nucleic acid that enhances expression of the desiredprotein (e.g., a plasmid encoding the desired protein).

Various exemplary embodiments of the cationic lipids of the presentinvention, lipid particles and compositions comprising the same, andtheir use to deliver active or therapeutic agents such as nucleic acidsto modulate gene and protein expression, are described in further detailbelow.

II. Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

The term “interfering RNA” or “RNAi” or “interfering RNA sequence” asused herein includes single-stranded RNA (e.g., mature miRNA, ssRNAioligonucleotides, ssDNAi oligonucleotides), double-stranded RNA (i.e.,duplex RNA such as siRNA, Dicer-substrate dsRNA, shRNA, aiRNA, orpre-miRNA), a DNA-RNA hybrid (see, e.g., PCT Publication No. WO2004/078941), or a DNA-DNA hybrid (see, e.g., PCT Publication No. WO2004/104199) that is capable of reducing or inhibiting the expression ofa target gene or sequence (e.g., by mediating the degradation orinhibiting the translation of mRNAs which are complementary to theinterfering RNA sequence) when the interfering RNA is in the same cellas the target gene or sequence. Interfering RNA thus refers to thesingle-stranded RNA that is complementary to a target mRNA sequence orto the double-stranded RNA formed by two complementary strands or by asingle, self-complementary strand. Interfering RNA may have substantialor complete identity to the target gene or sequence, or may comprise aregion of mismatch (i.e., a mismatch motif). The sequence of theinterfering RNA can correspond to the full-length target gene, or asubsequence thereof. Preferably, the interfering RNA molecules arechemically synthesized. The disclosures of each of the above patentdocuments are herein incorporated by reference in their entirety for allpurposes.

Interfering RNA includes “small-interfering RNA” or “siRNA,” e.g.,interfering RNA of about 15-60, 15-50, or 15-40 (duplex) nucleotides inlength, more typically about 15-30, 15-25, or 19-25 (duplex) nucleotidesin length, and is preferably about 20-24, 21-22, or 21-23 (duplex)nucleotides in length (e.g., each complementary sequence of thedouble-stranded siRNA is 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25nucleotides in length, preferably about 20-24, 21-22, or 21-23nucleotides in length, and the double-stranded siRNA is about 15-60,15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length, preferablyabout 18-22, 19-20, or 19-21 base pairs in length). siRNA duplexes maycomprise 3′ overhangs of about 1 to about 4 nucleotides or about 2 toabout 3 nucleotides and 5′ phosphate termini. Examples of siRNA include,without limitation, a double-stranded polynucleotide molecule assembledfrom two separate stranded molecules, wherein one strand is the sensestrand and the other is the complementary antisense strand; adouble-stranded polynucleotide molecule assembled from a single strandedmolecule, where the sense and antisense regions are linked by a nucleicacid-based or non-nucleic acid-based linker; a double-strandedpolynucleotide molecule with a hairpin secondary structure havingself-complementary sense and antisense regions; and a circularsingle-stranded polynucleotide molecule with two or more loop structuresand a stem having self-complementary sense and antisense regions, wherethe circular polynucleotide can be processed in vivo or in vitro togenerate an active double-stranded siRNA molecule. As used herein, theterm “siRNA” includes RNA-RNA duplexes as well as DNA-RNA hybrids (see,e.g., PCT Publication No. WO 2004/078941).

Preferably, siRNA are chemically synthesized. siRNA can also begenerated by cleavage of longer dsRNA (e.g., dsRNA greater than about 25nucleotides in length) with the E. coli RNase III or Dicer. Theseenzymes process the dsRNA into biologically active siRNA (see, e.g.,Yang et al., Proc. Natl. Acad. Sci. USA, 99:9942-9947 (2002); Calegariet al., Proc. Natl. Acad. Sci. USA, 99:14236 (2002); Byrom et al.,Ambion TechNotes, 10(1):4-6 (2003); Kawasaki et al., Nucleic Acids Res.,31:981-987 (2003); Knight et al., Science, 293:2269-2271 (2001); andRobertson et al., J Biol. Chem., 243:82 (1968)). Preferably, dsRNA areat least 50 nucleotides to about 100, 200, 300, 400, or 500 nucleotidesin length. A dsRNA may be as long as 1000, 1500, 2000, 5000 nucleotidesin length, or longer. The dsRNA can encode for an entire gene transcriptor a partial gene transcript. In certain instances, siRNA may be encodedby a plasmid (e.g., transcribed as sequences that automatically foldinto duplexes with hairpin loops).

As used herein, the term “mismatch motif” or “mismatch region” refers toa portion of an interfering RNA (e.g., siRNA) sequence that does nothave 100% complementarity to its target sequence. An interfering RNA mayhave at least one, two, three, four, five, six, or more mismatchregions. The mismatch regions may be contiguous or may be separated by1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleotides. The mismatchmotifs or regions may comprise a single nucleotide or may comprise two,three, four, five, or more nucleotides.

The phrase “inhibiting expression of a target gene” refers to theability of a nucleic acid such as an interfering RNA (e.g., siRNA) tosilence, reduce, or inhibit the expression of a target gene. To examinethe extent of gene silencing, a test sample (e.g., a sample of cells inculture expressing the target gene) or a test mammal (e.g., a mammalsuch as a human or an animal model such as a rodent (e.g., mouse) or anon-human primate (e.g., monkey) model) is contacted with a nucleic acidsuch as an interfering RNA (e.g., siRNA) that silences, reduces, orinhibits expression of the target gene. Expression of the target gene inthe test sample or test animal is compared to expression of the targetgene in a control sample (e.g., a sample of cells in culture expressingthe target gene) or a control mammal (e.g., a mammal such as a human oran animal model such as a rodent (e.g., mouse) or non-human primate(e.g., monkey) model) that is not contacted with or administered thenucleic acid (e.g., interfering RNA). The expression of the target genein a control sample or a control mammal may be assigned a value of 100%.In particular embodiments, silencing, inhibition, or reduction ofexpression of a target gene is achieved when the level of target geneexpression in the test sample or the test mammal relative to the levelof target gene expression in the control sample or the control mammal isabout 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%,30%, 25%, 20%, 15%, 10%, 5%, or 0%. In other words, the nucleic acids(e.g., interfering RNAs) are capable of silencing, reducing, orinhibiting the expression of a target gene by at least about 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% in a test sample or a test mammal relative to thelevel of target gene expression in a control sample or a control mammalnot contacted with or administered the nucleic acid (e.g., interferingRNA). Suitable assays for determining the level of target geneexpression include, without limitation, examination of protein or mRNAlevels using techniques known to those of skill in the art, such as,e.g., dot blots, Northern blots, in situ hybridization, ELISA,immunoprecipitation, enzyme function, as well as phenotypic assays knownto those of skill in the art.

An “effective amount” or “therapeutically effective amount” of an activeagent or therapeutic agent such as a therapeutic nucleic acid (e.g.,interfering RNA such as an siRNA) is an amount sufficient to produce thedesired effect, e.g., an inhibition of expression of a target sequencein comparison to the normal expression level detected in the absence ofthe nucleic acid (e.g., interfering RNA). Inhibition of expression of atarget gene or target sequence is achieved when the value obtained witha nucleic acid such as an interfering RNA (e.g., siRNA) relative to thecontrol is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%,40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Suitable assays formeasuring expression of a target gene or target sequence include, e.g.,examination of protein or RNA levels using techniques known to those ofskill in the art such as dot blots, northern blots, in situhybridization, ELISA, immunoprecipitation, enzyme function, as well asphenotypic assays known to those of skill in the art.

By “decrease,” “decreasing,” “reduce,” or “reducing” of an immuneresponse by a nucleic acid such as an interfering RNA (e.g., siRNA) isintended to mean a detectable decrease of an immune response to a givennucleic acid (e.g., a modified interfering RNA). In some instances, theamount of decrease of an immune response by a nucleic acid such as amodified interfering RNA may be determined relative to the level of animmune response in the presence of an unmodified interfering RNA. As anon-limiting example, a detectable decrease can be about 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 100%, or more lower than the immune response detected in thepresence of the unmodified interfering RNA. A decrease in the immuneresponse to a nucleic acid (e.g., interfering RNA) is typically measuredby a decrease in cytokine production (e.g., IFNγ, IFNα, TNFα, IL-6,IL-8, or IL-12) by a responder cell in vitro or a decrease in cytokineproduction in the sera of a mammalian subject after administration ofthe nucleic acid (e.g., interfering RNA).

As used herein, the term “responder cell” refers to a cell, preferably amammalian cell, that produces a detectable immune response whencontacted with an immunostimulatory nucleic acid such as an unmodifiedinterfering RNA (e.g., unmodified siRNA). Exemplary responder cellsinclude, without limitation, dendritic cells, macrophages, peripheralblood mononuclear cells (PBMCs), splenocytes, and the like. Detectableimmune responses include, e.g., production of cytokines or growthfactors such as TNF-α, IFN-α, IFN-β, IFN-γ, IL-1, IL-2, IL-3, IL-4,IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, TGF, and combinations thereof.Detectable immune responses also include, e.g., induction ofinterferon-induced protein with tetratricopeptide repeats 1 (IFIT1)mRNA.

The term “nucleic acid” as used herein refers to a polymer containing atleast two deoxyribonucleotides or ribonucleotides in either single- ordouble-stranded form and includes DNA, RNA, and hybrids thereof. DNA maybe in the form of, e.g., antisense molecules, plasmid DNA, DNA-DNAduplexes, pre-condensed DNA, PCR products, vectors (P1, PAC, BAC, YAC,artificial chromosomes), expression cassettes, chimeric sequences,chromosomal DNA, or derivatives and combinations of these groups. RNAmay be in the form of small interfering RNA (siRNA), Dicer-substratedsRNA, small hairpin RNA (shRNA), asymmetrical interfering RNA (aiRNA),microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), andcombinations thereof. Nucleic acids include nucleic acids containingknown nucleotide analogs or modified backbone residues or linkages,which are synthetic, naturally occurring, and non-naturally occurring,and which have similar binding properties as the reference nucleic acid.Examples of such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2′-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unlessspecifically limited, the term encompasses nucleic acids containingknown analogues of natural nucleotides that have similar bindingproperties as the reference nucleic acid. Unless otherwise indicated, aparticular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions), alleles, orthologs, SNPs, and complementary sequences aswell as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res., 19:5081 (1991); Ohtsuka et al., J Biol. Chem., 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)).“Nucleotides” contain a sugar deoxyribose (DNA) or ribose (RNA), a base,and a phosphate group. Nucleotides are linked together through thephosphate groups. “Bases” include purines and pyrimidines, which furtherinclude natural compounds adenine, thymine, guanine, cytosine, uracil,inosine, and natural analogs, and synthetic derivatives of purines andpyrimidines, which include, but are not limited to, modifications whichplace new reactive groups such as, but not limited to, amines, alcohols,thiols, carboxylates, and alkylhalides.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises partial length or entire length coding sequencesnecessary for the production of a polypeptide or precursor polypeptide.

“Gene product,” as used herein, refers to a product of a gene such as anRNA transcript or a polypeptide.

The term “lipid” refers to a group of organic compounds that include,but are not limited to, esters of fatty acids and are characterized bybeing insoluble in water, but soluble in many organic solvents. They areusually divided into at least three classes: (1) “simple lipids,” whichinclude fats and oils as well as waxes; (2) “compound lipids,” whichinclude phospholipids and glycolipids; and (3) “derived lipids” such assteroids.

The term “lipid particle” includes a lipid formulation that can be usedto deliver an active agent or therapeutic agent, such as a nucleic acid(e.g., interfering RNA) to a target site of interest (e.g., cell,tissue, organ, tumor, and the like). In preferred embodiments, the lipidparticle of the invention is a nucleic acid-lipid particle, which istypically formed from a cationic lipid, a non-cationic lipid, andoptionally a conjugated lipid that prevents aggregation of the particle.In other preferred embodiments, the active agent or therapeutic agent,such as a nucleic acid (e.g., interfering RNA), may be encapsulated inthe lipid portion of the particle, thereby protecting it from enzymaticdegradation.

As used herein, the term “SNALP” refers to a stable nucleic acid-lipidparticle. A SNALP represents a particle made from lipids (e.g., acationic lipid, a non-cationic lipid, and optionally a conjugated lipidthat prevents aggregation of the particle), wherein the nucleic acid(e.g., an interfering RNA) is fully encapsulated within the lipid. Incertain instances, SNALP are extremely useful for systemic applications,as they can exhibit extended circulation lifetimes following intravenous(i.v.) injection, they can accumulate at distal sites (e.g., sitesphysically separated from the administration site), and they can mediatesilencing of target gene expression at these distal sites. The nucleicacid may be complexed with a condensing agent and encapsulated within aSNALP as set forth in PCT Publication No. WO 00/03683, the disclosure ofwhich is herein incorporated by reference in its entirety for allpurposes.

The lipid particles of the invention (e.g., SNALP) typically have a meandiameter of from about 30 nm to about 150 nm, from about 40 nm to about150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm,from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, fromabout 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm,and are substantially non-toxic. In addition, nucleic acids, whenpresent in the lipid particles of the present invention, are resistantin aqueous solution to degradation with a nuclease. Nucleic acid-lipidparticles and their method of preparation are disclosed in, e.g., U.S.Patent Publication Nos. 20040142025 and 20070042031, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes.

As used herein, “lipid encapsulated” can refer to a lipid particle thatprovides an active agent or therapeutic agent, such as a nucleic acid(e.g., an interfering RNA such as an siRNA), with full encapsulation,partial encapsulation, or both. In a preferred embodiment, the nucleicacid (e.g., interfering RNA) is fully encapsulated in the lipid particle(e.g., to form a SNALP or other nucleic acid-lipid particle).

The term “lipid conjugate” refers to a conjugated lipid that inhibitsaggregation of lipid particles. Such lipid conjugates include, but arenot limited to, PEG-lipid conjugates such as, e.g., PEG coupled todialkyloxypropyls (e.g., PEG-DAA conjugates), PEG coupled todiacylglycerols (e.g., PEG-DAG conjugates), PEG coupled to cholesterol,PEG coupled to phosphatidylethanolamines, and PEG conjugated toceramides (see, e.g., U.S. Pat. No. 5,885,613), cationic PEG lipids,polyoxazoline (POZ)-lipid conjugates (e.g., POZ-DAA conjugates; see,e.g., U.S. application Ser. No. 13/006,277, filed Jan. 13, 2011),polyamide oligomers (e.g., ATTA-lipid conjugates), and mixtures thereof.Additional examples of POZ-lipid conjugates are described in PCTPublication No. WO 2010/006282. PEG or POZ can be conjugated directly tothe lipid or may be linked to the lipid via a linker moiety. Any linkermoiety suitable for coupling the PEG or the POZ to a lipid can be usedincluding, e.g., non-ester containing linker moieties andester-containing linker moieties. In certain preferred embodiments,non-ester containing linker moieties, such as amides or carbamates, areused. The disclosures of each of the above patent documents are hereinincorporated by reference in their entirety for all purposes.

The term “amphipathic lipid” refers, in part, to any suitable materialwherein the hydrophobic portion of the lipid material orients into ahydrophobic phase, while the hydrophilic portion orients toward theaqueous phase. Hydrophilic characteristics derive from the presence ofpolar or charged groups such as carbohydrates, phosphate, carboxylic,sulfato, amino, sulfhydryl, nitro, hydroxyl, and other like groups.Hydrophobicity can be conferred by the inclusion of apolar groups thatinclude, but are not limited to, long-chain saturated and unsaturatedaliphatic hydrocarbon groups and such groups substituted by one or morearomatic, cycloaliphatic, or heterocyclic group(s). Examples ofamphipathic compounds include, but are not limited to, phospholipids,aminolipids, and sphingolipids.

Representative examples of phospholipids include, but are not limitedto, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine,lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,dioleoylphosphatidylcholine, distearoylphosphatidylcholine, anddilinoleoylphosphatidylcholine. Other compounds lacking in phosphorus,such as sphingolipid, glycosphingolipid families, diacylglycerols, andβ-acyloxyacids, are also within the group designated as amphipathiclipids. Additionally, the amphipathic lipids described above can bemixed with other lipids including triglycerides and sterols.

The term “neutral lipid” refers to any of a number of lipid species thatexist either in an uncharged or neutral zwitterionic form at a selectedpH. At physiological pH, such lipids include, for example,diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

The term “non-cationic lipid” refers to any amphipathic lipid as well asany other neutral lipid or anionic lipid.

The term “anionic lipid” refers to any lipid that is negatively chargedat physiological pH. These lipids include, but are not limited to,phosphatidylglycerols, cardiolipins, diacylphosphatidylserines,diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines,N-succinyl phosphatidylethanolamines,N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols,palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifyinggroups joined to neutral lipids.

The term “hydrophobic lipid” refers to compounds having apolar groupsthat include, but are not limited to, long-chain saturated andunsaturated aliphatic hydrocarbon groups and such groups optionallysubstituted by one or more aromatic, cycloaliphatic, or heterocyclicgroup(s). Suitable examples include, but are not limited to,diacylglycerol, dialkylglycerol, N—N-dialkylamino,1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

The term “fusogenic” refers to the ability of a lipid particle, such asa SNALP, to fuse with the membranes of a cell. The membranes can beeither the plasma membrane or membranes surrounding organelles, e.g.,endosome, nucleus, etc.

As used herein, the term “aqueous solution” refers to a compositioncomprising in whole, or in part, water.

As used herein, the term “organic lipid solution” refers to acomposition comprising in whole, or in part, an organic solvent having alipid.

“Distal site,” as used herein, refers to a physically separated site,which is not limited to an adjacent capillary bed, but includes sitesbroadly distributed throughout an organism.

“Serum-stable” in relation to nucleic acid-lipid particles such as SNALPmeans that the particle is not significantly degraded after exposure toa serum or nuclease assay that would significantly degrade free DNA orRNA. Suitable assays include, for example, a standard serum assay, aDNAse assay, or an RNAse assay.

“Systemic delivery,” as used herein, refers to delivery of lipidparticles that leads to a broad biodistribution of an active agent suchas an interfering RNA (e.g., siRNA) within an organism. Some techniquesof administration can lead to the systemic delivery of certain agents,but not others. Systemic delivery means that a useful, preferablytherapeutic, amount of an agent is exposed to most parts of the body. Toobtain broad biodistribution generally requires a blood lifetime suchthat the agent is not rapidly degraded or cleared (such as by first passorgans (liver, lung, etc.) or by rapid, nonspecific cell binding) beforereaching a disease site distal to the site of administration. Systemicdelivery of lipid particles can be by any means known in the artincluding, for example, intravenous, subcutaneous, and intraperitoneal.In a preferred embodiment, systemic delivery of lipid particles is byintravenous delivery.

“Local delivery,” as used herein, refers to delivery of an active agentsuch as an interfering RNA (e.g., siRNA) directly to a target sitewithin an organism. For example, an agent can be locally delivered bydirect injection into a disease site such as a tumor, other target sitesuch as a site of inflammation, or a target organ such as the liver,heart, pancreas, kidney, and the like.

The term “mammal” refers to any mammalian species such as a human,mouse, rat, dog, cat, hamster, guinea pig, rabbit, livestock, and thelike.

The term “cancer” refers to any member of a class of diseasescharacterized by the uncontrolled growth of aberrant cells. The termincludes all known cancers and neoplastic conditions, whethercharacterized as malignant, benign, soft tissue, or solid, and cancersof all stages and grades including pre- and post-metastatic cancers.Examples of different types of cancer include, but are not limited to,liver cancer, lung cancer, colon cancer, rectal cancer, anal cancer,bile duct cancer, small intestine cancer, stomach (gastric) cancer,esophageal cancer; gallbladder cancer, pancreatic cancer, appendixcancer, breast cancer, ovarian cancer; cervical cancer, prostate cancer,renal cancer (e.g., renal cell carcinoma), cancer of the central nervoussystem, glioblastoma, skin cancer, lymphomas, choriocarcinomas, head andneck cancers, osteogenic sarcomas, and blood cancers. Non-limitingexamples of specific types of liver cancer include hepatocellularcarcinoma (HCC), secondary liver cancer (e.g., caused by metastasis ofsome other non-liver cancer cell type), and hepatoblastoma. As usedherein, a “tumor” comprises one or more cancerous cells.

III. Novel Cationic Lipids

The present invention provides, inter alia, novel cationic (amino)lipids that can advantageously be used in the lipid particles describedherein for the in vitro and/or in vivo delivery of therapeutic agentssuch as nucleic acids to cells. The novel cationic lipids of theinvention have the structures set forth in Formulas I-III herein, andinclude the (R) and/or (S) enantiomers thereof.

In some embodiments, a lipid of the present invention comprises aracemic mixture. In other embodiments, a lipid of the present inventioncomprises a mixture of one or more diastereomers. In certainembodiments, a lipid of the present invention is enriched in oneenantiomer, such that the lipid comprises at least about 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, or 95% enantiomeric excess. In certain otherembodiments, a lipid of the present invention is enriched in onediastereomer, such that the lipid comprises at least about 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, or 95% diastereomeric excess. In certainadditional embodiments, a lipid of the present invention is chirallypure (e.g., comprises a single optical isomer). In further embodiments,a lipid of the present invention is enriched in one optical isomer(e.g., an optically active isomer), such that the lipid comprises atleast about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% isomericexcess. The present invention provides the synthesis of the cationiclipids of Formula I-III as a racemic mixture or in optically pure form.

The terms “cationic lipid” and “amino lipid” are used interchangeablyherein to include those lipids and salts thereof having one, two, three,or more fatty acid or fatty alkyl chains and a pH-titratable amino headgroup (e.g., an alkylamino or dialkylamino head group). The cationiclipid is typically protonated (i.e., positively charged) at a pH belowthe pK_(a) of the cationic lipid and is substantially neutral at a pHabove the pK_(a). The cationic lipids of the invention may also betermed titratable cationic lipids.

The term “salts” includes any anionic and cationic complex, such as thecomplex formed between a cationic lipid disclosed herein and one or moreanions. Non-limiting examples of anions include inorganic and organicanions, e.g., hydride, fluoride, chloride, bromide, iodide, oxalate(e.g., hemioxalate), phosphate, phosphonate, hydrogen phosphate,dihydrogen phosphate, oxide, carbonate, bicarbonate, nitrate, nitrite,nitride, bisulfite, sulfide, sulfite, bisulfate, sulfate, thiosulfate,hydrogen sulfate, borate, formate, acetate, benzoate, citrate, tartrate,lactate, acrylate, polyacrylate, fumarate, maleate, itaconate,glycolate, gluconate, malate, mandelate, tiglate, ascorbate, salicylate,polymethacrylate, perchlorate, chlorate, chlorite, hypochlorite,bromate, hypobromite, iodate, an alkylsulfonate, an arylsulfonate,arsenate, arsenite, chromate, dichromate, cyanide, cyanate, thiocyanate,hydroxide, peroxide, permanganate, and mixtures thereof. In particularembodiments, the salts of the cationic lipids disclosed herein arecrystalline salts.

The term “alkyl” includes a straight chain or branched, noncyclic orcyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbonatoms. Representative saturated straight chain alkyls include, but arenot limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, andthe like, while saturated branched alkyls include, without limitation,isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.Representative saturated cyclic alkyls include, but are not limited to,the C₃₋₈ cycloalkyls described herein, while unsaturated cyclic alkylsinclude, without limitation, the C₃₋₈ cycloalkenyls described herein.

The term “heteroalkyl,” includes a straight chain or branched, noncyclicor cyclic, saturated aliphatic hydrocarbon as defined above having fromabout 1 to about 5 heteroatoms (i.e., 1, 2, 3, 4, or 5 heteroatoms) suchas, for example, O, N, Si, and/or S, wherein the nitrogen and sulfuratoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroalkyl group can be attached to theremainder of the molecule through a carbon atom or a heteroatom.

The term “cyclic alkyl” includes any of the substituted or unsubstitutedcycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenylgroups described below.

The term “cycloalkyl” includes a substituted or unsubstituted cyclicalkyl group having from about 3 to about 8 carbon atoms (i.e., 3, 4, 5,6, 7, or 8 carbon atoms) as ring vertices. Preferred cycloalkyl groupsinclude those having from about 3 to about 6 carbon atoms as ringvertices. Examples of C₃₋₈ cycloalkyl groups include, but are notlimited to, cyclopropyl, methyl-cyclopropyl, dimethyl-cyclopropyl,cyclobutyl, methyl-cyclobutyl, cyclopentyl, methyl-cyclopentyl,cyclohexyl, methyl-cyclohexyl, dimethyl-cyclohexyl, cycloheptyl, andcyclooctyl, as well as other substituted C₃₋₈ cycloalkyl groups.

The term “heterocycloalkyl” includes a substituted or unsubstitutedcyclic alkyl group as defined above having from about 1 to about 3heteroatoms as ring members selected from the group consisting of O, N,Si and S, wherein the nitrogen and sulfur atoms may optionally beoxidized and the nitrogen heteroatom may optionally be quaternized. Theheterocycloalkyl group can be attached to the remainder of the moleculethrough a carbon atom or a heteroatom.

The term “cycloalkenyl” includes a substituted or unsubstituted cyclicalkenyl group having from about 3 to about 8 carbon atoms (i.e., 3, 4,5, 6, 7, or 8 carbon atoms) as ring vertices. Preferred cycloalkenylgroups are those having from about 3 to about 6 carbon atoms as ringvertices. Examples of C₃₋₈ cycloalkenyl groups include, but are notlimited to, cyclopropenyl, methyl-cyclopropenyl, dimethyl-cyclopropenyl,cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, andcyclooctenyl, as well as other substituted C₃₋₈ cycloalkenyl groups.

The term “heterocycloalkenyl” includes a substituted or unsubstitutedcyclic alkenyl group as defined above having from about 1 to about 3heteroatoms as ring members selected from the group consisting of O, N,Si and S, wherein the nitrogen and sulfur atoms may optionally beoxidized and the nitrogen heteroatom may optionally be quaternized. Theheterocycloalkenyl group can be attached to the remainder of themolecule through a carbon atom or a heteroatom.

The term “alkoxy” includes a group of the formula alkyl-O—, wherein“alkyl” has the previously given definition. Non-limiting examples ofalkoxy groups include methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy,iso-butoxy, sec-butoxy and tert-butoxy.

The term “alkenyl” includes an alkyl, as defined above, containing atleast one double bond between adjacent carbon atoms. Alkenyls includeboth cis and trans isomers. Representative straight chain and branchedalkenyls include, but are not limited to, ethylenyl, propylenyl,1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl,3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and thelike. Representative cyclic alkenyls are described above.

The term “alkynyl” includes any alkyl or alkenyl, as defined above,which additionally contains at least one triple bond between adjacentcarbons. Representative straight chain and branched alkynyls include,without limitation, acetylenyl, propynyl, 1-butynyl, 2-butynyl,1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.

The term “aryl” includes a polyunsaturated, typically aromatic,hydrocarbon group which can be a single ring or multiple rings (up tothree rings) which are fused together or linked covalently, and whichoptionally carries one or more substituents, such as, for example,halogen, trifluoromethyl, amino, alkyl, alkoxy, alkylcarbonyl, cyano,carbamoyl, alkoxycarbamoyl, methylendioxy, carboxy, alkoxycarbonyl,aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, hydroxy, nitro,and the like. Non-limiting examples of unsubstituted aryl groups includephenyl, naphthyl, and biphenyl. Examples of substituted aryl groupsinclude, but are not limited to, phenyl, chlorophenyl,trifluoromethylphenyl, chlorofluorophenyl, and aminophenyl.

The terms “alkylthio,” “alkylsulfonyl,” “alkylsulfinyl,” and“arylsulfonyl” include groups having the formula —S—R^(i), —S(O)₂—R^(i),—S(O)—R^(i) and —S(O)₂R^(j), respectively, wherein R^(i) is an alkylgroup as previously defined and R^(j) is an aryl group as previouslydefined.

The terms “alkenyloxy” and “alkynyloxy” include groups having theformula —O—R^(i), wherein R^(i) is an alkenyl or alkynyl group,respectively.

The terms “alkenylthio” and “alkynylthio” include groups having theformula —S—R^(k), wherein R^(k) is an alkenyl or alkynyl group,respectively.

The term “alkoxycarbonyl” includes a group having the formula—C(O)O—R^(i), wherein R^(i) is an alkyl group as defined above andwherein the total number of carbon atoms refers to the combined alkyland carbonyl moieties.

The term “acyl” includes any alkyl, alkenyl, or alkynyl wherein thecarbon at the point of attachment is substituted with an oxo group, asdefined below. The following are non-limiting examples of acyl groups:—C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl.

The term “heterocycle” includes a 5- to 7-membered monocyclic, or 7- to10-membered bicyclic, heterocyclic ring which is either saturated,unsaturated, or aromatic, and which contains one, two, three, or moreheteroatoms independently selected from nitrogen (N), oxygen (O), andsulfur (S), and wherein the nitrogen and sulfur heteroatoms may beoptionally oxidized, and the nitrogen heteroatom may be optionallyquaternized, including bicyclic rings in which any of the aboveheterocycles are fused to a benzene ring. The heterocycle may beattached via any heteroatom or carbon atom. Heterocycles include, butare not limited to, heteroaryls as defined below, as well asmorpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl,hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The term “heteroaryl” includes an aromatic 5- to 10-membered heterocyclewhich contains one, two, three, or more heteroatoms selected fromnitrogen (N), oxygen (O), and sulfur (S). The heteroaryl can besubstituted on one or more carbon atoms with substituents such as, forexample, halogen, alkyl, alkoxy, cyano, haloalkyl (e.g.,trifluoromethyl), heterocyclyl (e.g., morpholinyl or pyrrolidinyl), andthe like. Non-limiting examples of heteroaryls include pyridinyl andfuranyl.

The term “halogen” includes fluoro, chloro, bromo, and iodo.

The terms “optionally substituted alkyl,” “optionally substituted cyclicalkyl,” “optionally substituted alkenyl,” “optionally substitutedalkynyl,” “optionally substituted acyl,” and “optionally substitutedheterocycle” mean that, when substituted, at least one hydrogen atom isreplaced with a substituent. In the case of an “oxo” substituent (═O),two hydrogen atoms are replaced. Non-limiting examples of substituentsinclude oxo, halogen, heterocycle, —CN, —OR^(x), —NR^(x)R^(y),—NR^(x)C(═O)R^(y), —NR^(x)SO₂R^(y), —C(═O)R, —C(═O)OR^(x),—C(═O)NR^(x)R^(y), —SO_(n)R^(x), and —SO_(n)NR^(x)R^(y), wherein n is 0,1, or 2, R^(x) and R^(y) are the same or different and are independentlyhydrogen, alkyl, or heterocycle, and each of the alkyl and heterocyclesubstituents may be further substituted with one or more of oxo,halogen, —OH, —CN, alkyl, —OR^(x), heterocycle, —NR^(x)R^(y),—NR^(x)C(═O)R^(y), —NR^(x)SO₂R^(y), —C(═O)R^(x), —C(═O)OR^(x),—C(═O)NR^(x)R^(y), —SO_(n)R^(x), and —SO_(n)NR^(x)R^(y). The term“optionally substituted,” when used before a list of substituents, meansthat each of the substituents in the list may be optionally substitutedas described herein.

In one aspect, the present invention provides a cationic lipid ofFormula I having the following structure:

or salts thereof, wherein:

-   -   R¹ and R² are either the same or different and are independently        hydrogen (H) or an optionally substituted C₁-C₆ alkyl, C₂-C₆        alkenyl, or C₂-C₆ alkynyl, or R¹ and R² may join to form an        optionally substituted heterocyclic ring;    -   R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to        provide a quaternary amine;    -   R⁴ and R⁵ are either the same or different and are independently        an optionally substituted C₁₀-C₂₄ alkyl, C₁₀-C₂₄ alkenyl,        C₁₀-C₂₄ alkynyl, or C₁₀-C₂₄ acyl, wherein at least one of R⁴ and        R⁵ comprises at least 1, 2, 3, 4, 5, 6, or more optionally        substituted cyclic alkyl groups (e.g., 1-2, 1-3, 1-4, 1-5, 1-6,        2-3, 2-4, 2-5, or 2-6 optionally substituted cyclic alkyl        groups);    -   X and Y are either the same or different and are independently        O, S, N(R⁶), C(O), C(O)O, OC(O), C(O)N(R⁶), N(R⁶)C(O),        OC(O)N(R⁶), N(R⁶)C(O)O, C(O)S, C(S)O, S(O), S(O)(O), C(S), or an        optionally substituted heterocyclic ring, wherein R⁶ is        hydrogen (H) or an optionally substituted C₁-C₁₀ alkyl, C₂-C₁₀        alkenyl, or C₂-C₁₀ alkynyl; and    -   Z is either absent or is an optionally substituted C₁-C₆ alkyl,        C₂-C₆ alkenyl, or C₂-C₆ alkynyl.

In some embodiments, R¹ and R² are each independently hydrogen (H) or anoptionally substituted C₁-C₂ alkyl, C₁-C₃ alkyl, C₁-C₄ alkyl, C₁-C₅alkyl, C₂-C₃ alkyl, C₂-C₄ alkyl, C₂-C₅ alkyl, C₂-C₆ alkyl, C₃-C₄ alkyl,C₃-C₅ alkyl, C₃-C₆ alkyl, C₄-C₅ alkyl, C₄-C₆ alkyl, C₅-C₆ alkyl, C₂-C₃alkenyl, C₂-C₄ alkenyl, C₂-C₅ alkenyl, C₂-C₆ alkenyl, C₃-C₄ alkenyl,C₃-C₅ alkenyl, C₃-C₆ alkenyl, C₄-C₅ alkenyl, C₄-C₆ alkenyl, C₅-C₆alkenyl, C₂-C₃ alkynyl, C₂-C₄ alkynyl, C₂-C₅ alkynyl, C₂-C₆ alkynyl,C₃-C₄ alkynyl, C₃-C₅ alkynyl, C₃-C₆ alkynyl, C₄-C₅ alkynyl, C₄-C₆alkynyl, or C₅-C₆ alkynyl. In particular embodiments, R¹ and R² are bothmethyl groups, both ethyl groups, or a combination of one methyl groupand one ethyl group.

In certain instances, R³ is absent when the pH is above the pK_(a) ofthe cationic lipid and R³ is hydrogen (H) when the pH is below thepK_(a) of the cationic lipid such that the amino head group isprotonated. In certain other instances, R³ is an optionally substitutedC₁-C₂ alkyl, C₁-C₃ alkyl, C₁-C₄ alkyl, C₁-C₅ alkyl, C₂-C₃ alkyl, C₂-C₄alkyl, C₂-C₅ alkyl, C₂-C₆ alkyl, C₃-C₄ alkyl, C₃-C₅ alkyl, C₃-C₆ alkyl,C₄-C₅ alkyl, C₄-C₆ alkyl, or C₅-C₆ alkyl to provide a quaternary amine.

In certain embodiments, R¹ and R² are joined to form an optionallysubstituted heterocyclic ring comprising 1, 2, 3, 4, 5, 6, or morecarbon atoms and 1, 2, 3, 4, or more heteroatoms such as nitrogen (N),oxygen (O), sulfur (S), and mixtures thereof. In some embodiments, theoptionally substituted heterocyclic ring comprises from 2 to 5 carbonatoms and from 1 to 3 heteroatoms such as nitrogen (N), oxygen (O),and/or sulfur (S). In certain embodiments, the heterocyclic ringcomprises an optionally substituted imidazole, triazole (e.g.,1,2,3-triazole, 1,2,4-triazole), pyrazole, thiazole, pyrrole, furan,oxazole, isoxazole, oxazoline, oxazolidine, oxadiazole, tetrazole, andthe like. In some instances, the optionally substituted heterocyclicring comprises 5 carbon atoms and 1 nitrogen atom, wherein theheterocyclic ring can be substituted with a substituent such as ahydroxyl (—OH) group at the ortho, meta, and/or para positions. Incertain instances, the heterocyclic ring comprises an optionallysubstituted imidazole group.

In other embodiments, R⁶ is hydrogen (H) or an optionally substitutedC₁-C₂ alkyl, C₁-C₃ alkyl, C₁-C₄ alkyl, C₁-C₅ alkyl, C₁-C₆ alkyl, C₁-C₇alkyl, C₁-C₈ alkyl, C₁-C₉ alkyl, C₂-C₃ alkyl, C₂-C₄ alkyl, C₂-C₅ alkyl,C₂-C₆ alkyl, C₂-C₇ alkyl, C₂-C₈ alkyl, C₂-C₉ alkyl, C₂-C₁₀ alkyl, C₃-C₄alkyl, C₃-C₅ alkyl, C₃-C₆ alkyl, C₃-C₇ alkyl, C₃-C₈ alkyl, C₃-C₉ alkyl,C₃-C₁₀ alkyl, C₄-C₅ alkyl, C₄-C₆ alkyl, C₄-C₇ alkyl, C₄-C₈ alkyl, C₄-C₉alkyl, C₄-C₁₀ alkyl, C₅-C₆ alkyl, C₅-C₇ alkyl, C₅-C₈ alkyl, C₅-C₉ alkyl,C₅-C₁₀ alkyl, C₆-C₇ alkyl, C₆-C₈ alkyl, C₆-C₉ alkyl, C₆-C₁₀ alkyl, C₇-C₈alkyl, C₇-C₉ alkyl, C₇-C₁₀ alkyl, C₈-C₉ alkyl, C₈-C₁₀ alkyl, C₉-C₁₀alkyl, C₂-C₃ alkenyl, C₂-C₄ alkenyl, C₂-C₅ alkenyl, C₂-C₆ alkenyl, C₂-C₇alkenyl, C₂-C₈ alkenyl, C₂-C₉ alkenyl, C₃-C₄ alkenyl, C₃-C₅ alkenyl,C₃-C₆ alkenyl, C₃-C₇ alkenyl, C₃-C₈ alkenyl, C₃-C₉ alkenyl, C₃-C₁₀alkenyl, C₄-C₅ alkenyl, C₄-C₆ alkenyl, C₄-C₇ alkenyl, C₄-C₈ alkenyl,C₄-C₉ alkenyl, C₄-C₁₀ alkenyl, C₅-C₆ alkenyl, C₅-C₇ alkenyl, C₅-C₈alkenyl, C₅-C₉ alkenyl, C₅-C₁₀ alkenyl, C₆-C₇ alkenyl, C₆-C₈ alkenyl,C₆-C₉ alkenyl, C₆-C₁₀ alkenyl, C₇-C₈ alkenyl, C₇-C₉ alkenyl, C₇-C₁₀alkenyl, C₈-C₉ alkenyl, C₈-C₁₀ alkenyl, C₉-C₁₀ alkenyl, C₂-C₃ alkynyl,C₂-C₄ alkynyl, C₂-C₅ alkynyl, C₂-C₆ alkynyl, C₂-C₇ alkynyl, C₂-C₈alkynyl, C₂-C₉ alkynyl, C₃-C₄ alkynyl, C₃-C₅ alkynyl, C₃-C₆ alkynyl,C₃-C₇ alkynyl, C₃-C₈ alkynyl, C₃-C₉ alkynyl, C₃-C₁₀ alkynyl, C₄-C₅alkynyl, C₄-C₆ alkynyl, C₄-C₇ alkynyl, C₄-C₈ alkynyl, C₄-C₉ alkynyl,C₄-C₁₀ alkynyl, C₅-C₆ alkynyl, C₅-C₇ alkynyl, C₅-C₈ alkynyl, C₅-C₉alkynyl, C₅-C₁₀ alkynyl, C₆-C₇ alkynyl, C₆-C₈ alkynyl, C₆-C₉ alkynyl,C₆-C₁₀ alkynyl, C₇-C₈ alkynyl, C₇-C₉ alkynyl, C₇-C₁₀ alkynyl, C₈-C₉alkynyl, C₈-C₁₀ alkynyl, or C₉-C₁₀ alkynyl. In one particularembodiment, R⁶ is selected from the group consisting of hydrogen (H), aC₁ alkyl (methyl) group, and a C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, and C₁₀alkyl, alkenyl, and alkynyl group.

In one embodiment, X and Y are independently selected from the groupconsisting of O, C(O)O, C(O)N(R⁶), N(R⁶)C(O)O, and C(O)S. In oneparticular embodiment, X and Y are both oxygen (O). In anotherparticular embodiment, at least one of (e.g., both) X and Y isN(R⁶)C(O)O and each R⁶ is independently hydrogen (H), a methyl group, ora C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, or C₁₀ alkyl, alkenyl, or alkynylgroup. In certain other embodiments, at least one of (e.g., both) X andY is an independently selected optionally substituted heterocyclic ring.In particular embodiments, the heterocyclic ring comprises 1, 2, 3, 4,5, 6, or more carbon atoms and 1, 2, 3, 4, or more heteroatoms such asnitrogen (N), oxygen (O), sulfur (S), and mixtures thereof. In someembodiments, the optionally substituted heterocyclic ring comprises from2 to 5 carbon atoms and from 1 to 3 heteroatoms such as nitrogen (N),oxygen (O), and/or sulfur (S). In certain embodiments, the heterocyclicring comprises an optionally substituted imidazole, triazole (e.g.,1,2,3-triazole, 1,2,4-triazole), pyrazole, thiazole, pyrrole, furan,oxazole, isoxazole, oxazoline, oxazolidine, oxadiazole, tetrazole, andthe like.

In certain embodiments, R⁴ and R⁵ are independently an optionallysubstituted C₁₂-C₂₄, C₁₂-C₂₂, C₁₂-C₂₀, C₁₄-C₂₄, C₁₄-C₂₂, C₁₄-C₂₀,C₁₆-C₂₄, C₁₆-C₂₂, or C₁₆-C₂₀ alkyl, alkenyl, alkynyl, or acyl group(i.e., a C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, orC₂₄ alkyl, alkenyl, alkynyl, or acyl group), wherein at least one ormore of (e.g., both) R⁴ and R⁵ independently comprises at least 1, 2, 3,4, 5, or 6 optionally substituted cyclic alkyl groups (e.g., 1-2, 1-3,1-4, 1-5, 1-6, 2-3, 2-4, 2-5, or 2-6 optionally substituted cyclic alkylgroups). In particular embodiments, the at least 1, 2, 3, 4, 5, or 6optionally substituted cyclic alkyl groups present in R⁴ and/or R⁵ areindependently selected from the group consisting of an optionallysubstituted saturated cyclic alkyl group, an optionally substitutedunsaturated cyclic alkyl group, and combinations thereof. In certaininstances, the optionally substituted saturated cyclic alkyl groupcomprises an optionally substituted C₃₋₈ cycloalkyl group (e.g.,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, etc.). In preferred embodiments, the optionally substitutedsaturated cyclic alkyl group comprises a cyclopropyl group, optionallycontaining one or more substituents and/or heteroatoms. In otherinstances, the optionally substituted unsaturated cyclic alkyl groupcomprises an optionally substituted C₃₋₈ cycloalkenyl group (e.g.,cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl,cyclooctenyl, etc.). In particular embodiments, R⁴ and R⁵ are the samelength (e.g., C₁₂-C₂₀ or C₁₈ alkyl chains) and contain the same numberand type of cyclic alkyl group (e.g., one, two, or three C₃₋₈ cycloalkylgroups such as one, two, or three cyclopropyl groups on each of R⁴ andR⁵).

In certain other embodiments, one of R⁴ or R⁵ comprises at least 1, 2,3, 4, 5, or 6 sites of unsaturation (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 2-3,2-4, 2-5, or 2-6 sites of unsaturation) or a substituted alkyl or acylgroup, and the other side-chain comprises at least 1, 2, 3, 4, 5, or 6optionally substituted cyclic alkyl groups (e.g., 1-2, 1-3, 1-4, 1-5,1-6, 2-3, 2-4, 2-5, or 2-6 optionally substituted cyclic alkyl groups).In embodiments where one of R⁴ or R⁵ comprises at least 1, 2, 3, 4, 5,or 6 sites of unsaturation, the unsaturated side-chain may comprise adodecenyl moiety, a tetradecenyl (e.g., myristoleyl) moiety, ahexadecenyl (e.g., palmitoleyl) moiety, an octadecenyl (e.g., oleyl)moiety, an icosenyl moiety, a dodecadienyl moiety, a tetradecadienylmoiety, a hexadecadienyl moiety, an octadecadienyl moiety, anicosadienyl moiety, a dodecatrienyl moiety, a tetradectrienyl moiety, ahexadecatrienyl moiety, an octadecatrienyl moiety, an icosatrienylmoiety, or an acyl derivative thereof (e.g., oleoyl, linoleoyl,linolenoyl, γ-linolenoyl, etc.). In some instances, the octadecadienylmoiety is a linoleyl moiety. In other instances, the octadecatrienylmoiety is a linolenyl moiety or a γ-linolenyl moiety. In embodimentswhere one of R⁴ or R⁵ comprises a branched alkyl or acyl group (e.g., asubstituted alkyl or acyl group), the branched alkyl or acyl group maycomprise a C₁₂-C₂₄ alkyl or acyl having at least 1-6 (e.g., 1, 2, 3, 4,5, 6, or more) C₁-C₆ alkyl substituents. In particular embodiments, thebranched alkyl or acyl group comprises a C₁₂-C₂₀ or C₁₄-C₂₂ alkyl oracyl with 1-6 (e.g., 1, 2, 3, 4, 5, 6) C₁-C₄ alkyl (e.g., methyl, ethyl,propyl, or butyl) substituents. In some embodiments, the branched alkylgroup comprises a phytanyl (3,7,11,15-tetramethyl-hexadecanyl) moietyand the branched acyl group comprises a phytanoyl(3,7,11,15-tetramethyl-hexadecanoyl) moiety.

In particular embodiments, R⁴ and R⁵ are both independently selectedC₁₂-C₂₀ alkyl groups (i.e., C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, orC₂₀ alkyl groups) having at least 1, 2, 3, 4, 5, or 6 optionallysubstituted cyclic alkyl groups (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 2-3,2-4, 2-5, or 2-6 optionally substituted cyclic alkyl groups). Inpreferred embodiments, R⁴ and R⁵ are both C₁₈ alkyl groups having atleast one, two, three, or more optionally substituted cyclic alkylgroups such as, for example, an optionally substituted C₃₋₈ cycloalkylgroup (e.g., a cyclopropyl group, optionally containing one or moresubstituents and/or heteroatoms). In certain embodiments, each of theoptionally substituted cyclic alkyl groups is independently selected andcan be the same cyclic alkyl group (e.g., all cyclopropyl groups) ordifferent cyclic alkyl groups (e.g., cyclopropyl and other cycloalkyl,heterocycloalkyl, cycloalkenyl, and/or heterocycloalkenyl groups).

In preferred embodiments, the optionally substituted cyclic alkyl groupspresent in R⁴ and/or R⁵ are located at the site(s) of unsaturation inthe corresponding unsaturated side-chain. As a non-limiting example, oneor both of R⁴ and R⁵ are C₁₈ alkyl groups having 1, 2, or 3 optionallysubstituted cyclic alkyl groups, wherein the optionally substitutedcyclic alkyl groups (e.g., independently selected cyclopropyl groups)are located at one or more (e.g., all) of the sites of unsaturationpresent in a corresponding linoleyl moiety, linolenyl moiety, orγ-linolenyl moiety.

In alternative embodiments to the cationic lipid of Formula I, R⁴ and R⁵are different and are independently an optionally substituted C₁-C₂₄alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, or C₁-C₂₄ acyl, wherein at leastone of R⁴ and R⁵ comprises at least one optionally substituted cyclicalkyl group. In certain embodiments, R⁴ and R⁵ are different and areindependently an optionally substituted C₄-C₂₀ alkyl, C₄-C₂₀ alkenyl,C₄-C₂₀ alkynyl, or C₄-C₂₀ acyl. In some instances, R⁴ is an optionallysubstituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄ alkenyl, C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄acyl, and R⁵ is an optionally substituted C₄-C₁₀ alkyl, C₄-C₁₀ alkenyl,C₄-C₁₀ alkynyl, or C₄-C₁₀ acyl. In other instances, R⁴ is an optionallysubstituted C₁₂-C₂₀ or C₁₄-C₂₂ alkyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkenyl,C₁₂-C₂₀ or C₁₄-C₂₂ alkynyl, or C₁₂-C₂₀ or C₁₄-C₂₂ acyl, and R⁵ is anoptionally substituted C₄-C₈ or C₆ alkyl, C₄-C₈ or C₆ alkenyl, C₄-C₈ orC₆ alkynyl, or C₄-C₈ or C₆ acyl. In certain instances, R⁴ is anoptionally substituted C₄-C₁₀ alkyl, C₄-C₁₀ alkenyl, C₄-C₁₀ alkynyl, orC₄-C₁₀ acyl, and R⁵ is an optionally substituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄alkenyl, C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl. In certain other instances,R⁴ is an optionally substituted C₄-C₈ or C₆ alkyl, C₄-C₈ or C₆ alkenyl,C₄-C₈ or C₆ alkynyl, or C₄-C₈ or C₆ acyl, and R⁵ is an optionallysubstituted C₁₂-C₂₀ or C₁₄-C₂₂ alkyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkenyl,C₁₂-C₂₀ or C₁₄-C₂₂ alkynyl, or C₁₂-C₂₀ or C₁₄-C₂₂ acyl. In particularembodiments, one or more of the optionally substituted cyclic alkylgroups present in R⁴ and/or R⁵ are as described above.

In some groups of embodiments to the cationic lipid of Formula I, R⁴ andR⁵ are either the same or different and are independently selected fromthe group consisting of:

In other groups of embodiments to the cationic lipid of Formula I, oneof R⁴ or R⁵ is selected from the group consisting of:

and the other of R⁴ or R⁵ is selected from the group consisting of:

In certain embodiments, Z is an optionally substituted C₁-C₂ alkyl,C₁-C₃ alkyl, C₁-C₄ alkyl, C₁-C₅ alkyl, C₂-C₃ alkyl, C₂-C₄ alkyl, C₂-C₅alkyl, C₂-C₆ alkyl, C₃-C₄ alkyl, C₃-C₅ alkyl, C₃-C₆ alkyl, C₄-C₅ alkyl,C₄-C₆ alkyl, C₅-C₆ alkyl, C₂-C₃ alkenyl, C₂-C₄ alkenyl, C₂-C₅ alkenyl,C₂-C₆ alkenyl, C₃-C₄ alkenyl, C₃-C₅ alkenyl, C₃-C₆ alkenyl, C₄-C₅alkenyl, C₄-C₆ alkenyl, C₅-C₆ alkenyl, C₂-C₃ alkynyl, C₂-C₄ alkynyl,C₂-C₅ alkynyl, C₂-C₆ alkynyl, C₃-C₄ alkynyl, C₃-C₅ alkynyl, C₃-C₆alkynyl, C₄-C₅ alkynyl, C₄-C₆ alkynyl, or C₅-C₆ alkynyl. In oneparticular embodiment, Z is (CH₂)_(n) and n is 0, 1, 2, 3, 4, 5, or 6(e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 2-3, 2-4, 2-5, or 2-6). In a preferredembodiment, n is 1. In certain other embodiments, n is 2 or 3.

In particular embodiments, the cationic lipid of Formula I has thefollowing structure:

or salts thereof, wherein R¹, R², R³, R⁴, R⁵, X, Y, and n are the sameas described above.

In some embodiments, the cationic lipid of Formula I forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula I is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

In particularly preferred embodiments, the cationic lipid of Formula Ihas one of the following structures:

In another aspect, the present invention provides a cationic lipid ofFormula II having the following structure:

or salts thereof, wherein:

-   -   R¹ and R² are either the same or different and are independently        an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆        alkynyl, or R¹ and R² may join to form an optionally substituted        heterocyclic ring;    -   R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to        provide a quaternary amine;    -   R⁴ and R⁵ are either the same or different and are independently        an optionally substituted C₁₀-C₂₄ alkyl, C₁₀-C₂₄ alkenyl,        C₁₀-C₂₄ alkynyl, or C₁₀-C₂₄ acyl, wherein at least one of R⁴ and        R⁵ comprises at least 1, 2, 3, 4, 5, 6, or more optionally        substituted cyclic alkyl groups (e.g., 1-2, 1-3, 1-4, 1-5, 1-6,        2-3, 2-4, 2-5, or 2-6 optionally substituted cyclic alkyl        groups);    -   m, n, and p are either the same or different and are        independently either 0, 1, or 2, with the proviso that m, n, and        p are not simultaneously 0;    -   X and Y are either the same or different and are independently        O, S, N(R⁶), C(O), C(O)O, OC(O), C(O)N(R⁶), N(R⁶)C(O),        OC(O)N(R⁶), N(R⁶)C(O)O, C(O)S, C(S)O, S(O), S(O)(O), or C(S),        wherein R⁶ is hydrogen (H) or an optionally substituted C₁-C₁₀        alkyl, C₂-C₁₀ alkenyl, or C₂-C₁₀ alkynyl; and    -   Z is either absent or is an optionally substituted C₁-C₆ alkyl,        C₂-C₆ alkenyl, or C₂-C₆ alkynyl.

In some embodiments, R¹ and R² are each independently hydrogen (H) or anoptionally substituted C₁-C₂ alkyl, C₁-C₃ alkyl, C₁-C₄ alkyl, C₁-C₅alkyl, C₂-C₃ alkyl, C₂-C₄ alkyl, C₂-C₅ alkyl, C₂-C₆ alkyl, C₃-C₄ alkyl,C₃-C₅ alkyl, C₃-C₆ alkyl, C₄-C₅ alkyl, C₄-C₆ alkyl, C₅-C₆ alkyl, C₂-C₃alkenyl, C₂-C₄ alkenyl, C₂-C₅ alkenyl, C₂-C₆ alkenyl, C₃-C₄ alkenyl,C₃-C₅ alkenyl, C₃-C₆ alkenyl, C₄-C₅ alkenyl, C₄-C₆ alkenyl, C₅-C₆alkenyl, C₂-C₃ alkynyl, C₂-C₄ alkynyl, C₂-C₅ alkynyl, C₂-C₆ alkynyl,C₃-C₄ alkynyl, C₃-C₅ alkynyl, C₃-C₆ alkynyl, C₄-C₅ alkynyl, C₄-C₆alkynyl, or C₅-C₆ alkynyl. In particular embodiments, R¹ and R² are bothmethyl groups, both ethyl groups, or a combination of one methyl groupand one ethyl group. In certain instances, R³ is absent when the pH isabove the pK_(a) of the cationic lipid and R³ is hydrogen (H) when thepH is below the pK_(a) of the cationic lipid such that the amino headgroup is protonated. In certain other instances, R³ is an optionallysubstituted C₁-C₂ alkyl, C₁-C₃ alkyl, C₁-C₄ alkyl, C₁-C₅ alkyl, C₂-C₃alkyl, C₂-C₄ alkyl, C₂-C₅ alkyl, C₂-C₆ alkyl, C₃-C₄ alkyl, C₃-C₅ alkyl,C₃-C₆ alkyl, C₄-C₅ alkyl, C₄-C₆ alkyl, or C₅-C₆ alkyl to provide aquaternary amine.

In certain embodiments, R¹ and R² are joined to form an optionallysubstituted heterocyclic ring comprising 1, 2, 3, 4, 5, 6, or morecarbon atoms and 1, 2, 3, 4, or more heteroatoms such as nitrogen (N),oxygen (O), sulfur (S), and mixtures thereof. In some embodiments, theoptionally substituted heterocyclic ring comprises from 2 to 5 carbonatoms and from 1 to 3 heteroatoms such as nitrogen (N), oxygen (O),and/or sulfur (S). In certain embodiments, the heterocyclic ringcomprises an optionally substituted imidazole, triazole (e.g.,1,2,3-triazole, 1,2,4-triazole), pyrazole, thiazole, pyrrole, furan,oxazole, isoxazole, oxazoline, oxazolidine, oxadiazole, tetrazole, andthe like. In some instances, the optionally substituted heterocyclicring comprises 5 carbon atoms and 1 nitrogen atom, wherein theheterocyclic ring can be substituted with a substituent such as ahydroxyl (—OH) group at the ortho, meta, and/or para positions. Incertain instances, the heterocyclic ring comprises an optionallysubstituted imidazole group.

In other embodiments, R⁶ is hydrogen (H) or an optionally substitutedC₁-C₂ alkyl, C₁-C₃ alkyl, C₁-C₄ alkyl, C₁-C₅ alkyl, C₁-C₆ alkyl, C₁-C₇alkyl, C₁-C₈ alkyl, C₁-C₉ alkyl, C₂-C₃ alkyl, C₂-C₄ alkyl, C₂-C₅ alkyl,C₂-C₆ alkyl, C₂-C₇ alkyl, C₂-C₈ alkyl, C₂-C₉ alkyl, C₂-C₁₀ alkyl, C₃-C₄alkyl, C₃-C₅ alkyl, C₃-C₆ alkyl, C₃-C₇ alkyl, C₃-C₈ alkyl, C₃-C₉ alkyl,C₃-C₁₀ alkyl, C₄-C₅ alkyl, C₄-C₆ alkyl, C₄-C₇ alkyl, C₄-C₈ alkyl, C₄-C₉alkyl, C₄-C₁₀ alkyl, C₅-C₆ alkyl, C₅-C₇ alkyl, C₅-C₈ alkyl, C₅-C₉ alkyl,C₅-C₁₀ alkyl, C₆-C₇ alkyl, C₆-C₈ alkyl, C₆-C₉ alkyl, C₆-C₁₀ alkyl, C₇-C₈alkyl, C₇-C₉ alkyl, C₇-C₁₀ alkyl, C₈-C₉ alkyl, C₈-C₁₀ alkyl, C₉-C₁₀alkyl, C₂-C₃ alkenyl, C₂-C₄ alkenyl, C₂-C₅ alkenyl, C₂-C₆ alkenyl, C₂-C₇alkenyl, C₂-C₈ alkenyl, C₂-C₉ alkenyl, C₃-C₄ alkenyl, C₃-C₅ alkenyl,C₃-C₆ alkenyl, C₃-C₇ alkenyl, C₃-C₈ alkenyl, C₃-C₉ alkenyl, C₃-C₁₀alkenyl, C₄-C₅ alkenyl, C₄-C₆ alkenyl, C₄-C₇ alkenyl, C₄-C₈ alkenyl,C₄-C₉ alkenyl, C₄-C₁₀ alkenyl, C₅-C₆ alkenyl, C₅-C₇ alkenyl, C₅-C₈alkenyl, C₅-C₉ alkenyl, C₅-C₁₀ alkenyl, C₆-C₇ alkenyl, C₆-C₈ alkenyl,C₆-C₉ alkenyl, C₆-C₁₀ alkenyl, C₇-C₈ alkenyl, C₇-C₉ alkenyl, C₇-C₁₀alkenyl, C₈-C₉ alkenyl, C₈-C₁₀ alkenyl, C₉-C₁₀ alkenyl, C₂-C₃ alkynyl,C₂-C₄ alkynyl, C₂-C₅ alkynyl, C₂-C₆ alkynyl, C₂-C₇ alkynyl, C₂-C₈alkynyl, C₂-C₉ alkynyl, C₃-C₄ alkynyl, C₃-C₅ alkynyl, C₃-C₆ alkynyl,C₃-C₇ alkynyl, C₃-C₈ alkynyl, C₃-C₉ alkynyl, C₃-C₁₀ alkynyl, C₄-C₅alkynyl, C₄-C₆ alkynyl, C₄-C₇ alkynyl, C₄-C₈ alkynyl, C₄-C₉ alkynyl,C₄-C₁₀ alkynyl, C₅-C₆ alkynyl, C₅-C₇ alkynyl, C₅-C₈ alkynyl, C₅-C₉alkynyl, C₅-C₁₀ alkynyl, C₆-C₇ alkynyl, C₆-C₈ alkynyl, C₆-C₉ alkynyl,C₆-C₁₀ alkynyl, C₇-C₈ alkynyl, C₇-C₉ alkynyl, C₇-C₁₀ alkynyl, C₈-C₉alkynyl, C₈-C₁₀ alkynyl, or C₉-C₁₀ alkynyl. In one particularembodiment, R⁶ is selected from the group consisting of hydrogen (H), aC₁ alkyl (methyl) group, and a C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, and C₁₀alkyl, alkenyl, and alkynyl group.

In one embodiment, X and Y are independently selected from the groupconsisting of O, C(O)O, C(O)N(R⁶), N(R⁶)C(O)O, and C(O)S. In oneparticular embodiment, X and Y are both oxygen (O). In anotherparticular embodiment, at least one of (e.g., both) X and Y isN(R⁶)C(O)O and each R⁶ is independently hydrogen (H), a methyl group, ora C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, or C₁₀ alkyl, alkenyl, or alkynylgroup.

In certain embodiments, R⁴ and R⁵ are independently an optionallysubstituted C₁₂-C₂₄, C₁₂-C₂₂, C₁₂-C₂₄, C₁₄-C₂₄, C₁₄-C₂₂, C₁₄-C₂₀,C₁₆-C₂₄, C₁₆-C₂₂, or C₁₆-C₂₀ alkyl, alkenyl, alkynyl, or acyl group(i.e., a C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, orC₂₄ alkyl, alkenyl, alkynyl, or acyl group), wherein at least one ormore of (e.g., both) R⁴ and R⁵ independently comprises at least 1, 2, 3,4, 5, or 6 optionally substituted cyclic alkyl groups (e.g., 1-2, 1-3,1-4, 1-5, 1-6, 2-3, 2-4, 2-5, or 2-6 optionally substituted cyclic alkylgroups). In particular embodiments, the at least 1, 2, 3, 4, 5, or 6optionally substituted cyclic alkyl groups present in R⁴ and/or R⁵ areindependently selected from the group consisting of an optionallysubstituted saturated cyclic alkyl group, an optionally substitutedunsaturated cyclic alkyl group, and combinations thereof. In certaininstances, the optionally substituted saturated cyclic alkyl groupcomprises an optionally substituted C₃₋₈ cycloalkyl group (e.g.,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, etc.). In preferred embodiments, the optionally substitutedsaturated cyclic alkyl group comprises a cyclopropyl group, optionallycontaining one or more substituents and/or heteroatoms. In otherinstances, the optionally substituted unsaturated cyclic alkyl groupcomprises an optionally substituted C₃₋₈ cycloalkenyl group (e.g.,cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl,cyclooctenyl, etc.). In particular embodiments, R⁴ and R⁵ are the samelength (e.g., C₁₂-C₂₀ or C₁₈ alkyl chains) and contain the same numberand type of cyclic alkyl group (e.g., one, two, or three C₃₋₈ cycloalkylgroups such as one, two, or three cyclopropyl groups on each of R⁴ andR⁵).

In certain other embodiments, one of R⁴ or R⁵ comprises at least 1, 2,3, 4, 5, or 6 sites of unsaturation (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 2-3,2-4, 2-5, or 2-6 sites of unsaturation) or a substituted alkyl or acylgroup, and the other side-chain comprises at least 1, 2, 3, 4, 5, or 6optionally substituted cyclic alkyl groups (e.g., 1-2, 1-3, 1-4, 1-5,1-6, 2-3, 2-4, 2-5, or 2-6 optionally substituted cyclic alkyl groups).In embodiments where one of R⁴ or R⁵ comprises at least 1, 2, 3, 4, 5,or 6 sites of unsaturation, the unsaturated side-chain may comprise adodecenyl moiety, a tetradecenyl (e.g., myristoleyl) moiety, ahexadecenyl (e.g., palmitoleyl) moiety, an octadecenyl (e.g., oleyl)moiety, an icosenyl moiety, a dodecadienyl moiety, a tetradecadienylmoiety, a hexadecadienyl moiety, an octadecadienyl moiety, anicosadienyl moiety, a dodecatrienyl moiety, a tetradectrienyl moiety, ahexadecatrienyl moiety, an octadecatrienyl moiety, an icosatrienylmoiety, or an acyl derivative thereof (e.g., oleoyl, linoleoyl,linolenoyl, γ-linolenoyl, etc.). In some instances, the octadecadienylmoiety is a linoleyl moiety. In other instances, the octadecatrienylmoiety is a linolenyl moiety or a γ-linolenyl moiety. In embodimentswhere one of R⁴ or R⁵ comprises a branched alkyl or acyl group (e.g., asubstituted alkyl or acyl group), the branched alkyl or acyl group maycomprise a C₁₂-C₂₄ alkyl or acyl having at least 1-6 (e.g., 1, 2, 3, 4,5, 6, or more) C₁-C₆ alkyl substituents. In particular embodiments, thebranched alkyl or acyl group comprises a C₁₂-C₂₀ or C₁₄-C₂₂ alkyl oracyl with 1-6 (e.g., 1, 2, 3, 4, 5, 6) C₁-C₄ alkyl (e.g., methyl, ethyl,propyl, or butyl) substituents. In some embodiments, the branched alkylgroup comprises a phytanyl (3,7,11,15-tetramethyl-hexadecanyl) moietyand the branched acyl group comprises a phytanoyl(3,7,11,15-tetramethyl-hexadecanoyl) moiety.

In particular embodiments, R⁴ and R⁵ are both independently selectedC₁₂-C₂₀ alkyl groups (i.e., C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, orC₂₀ alkyl groups) having at least 1, 2, 3, 4, 5, or 6 optionallysubstituted cyclic alkyl groups (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 2-3,2-4, 2-5, or 2-6 optionally substituted cyclic alkyl groups). Inpreferred embodiments, R⁴ and R⁵ are both C₁₈ alkyl groups having atleast one, two, three, or more optionally substituted cyclic alkylgroups such as, for example, an optionally substituted C₃₋₈ cycloalkylgroup (e.g., a cyclopropyl group, optionally containing one or moresubstituents and/or heteroatoms). In certain embodiments, each of theoptionally substituted cyclic alkyl groups is independently selected andcan be the same cyclic alkyl group (e.g., all cyclopropyl groups) ordifferent cyclic alkyl groups (e.g., cyclopropyl and other cycloalkyl,heterocycloalkyl, cycloalkenyl, and/or heterocycloalkenyl groups).

In preferred embodiments, the optionally substituted cyclic alkyl groupspresent in R⁴ and/or R⁵ are located at the site(s) of unsaturation inthe corresponding unsaturated side-chain. As a non-limiting example, oneor both of R⁴ and R⁵ are C₁₈ alkyl groups having 1, 2, or 3 optionallysubstituted cyclic alkyl groups, wherein the optionally substitutedcyclic alkyl groups (e.g., independently selected cyclopropyl groups)are located at one or more (e.g., all) of the sites of unsaturationpresent in a corresponding linoleyl moiety, linolenyl moiety, orγ-linolenyl moiety.

In alternative embodiments to the cationic lipid of Formula II, R⁴ andR⁵ are different and are independently an optionally substituted C₁-C₂₄alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, or C₁-C₂₄ acyl, wherein at leastone of R⁴ and R⁵ comprises at least one optionally substituted cyclicalkyl group. In certain embodiments, R⁴ and R⁵ are different and areindependently an optionally substituted C₄-C₂₀ alkyl, C₄-C₂₀ alkenyl,C₄-C₂₀ alkynyl, or C₄-C₂₀ acyl. In some instances, R⁴ is an optionallysubstituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄ alkenyl, C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄acyl, and R⁵ is an optionally substituted C₄-C₁₀ alkyl, C₄-C₁₀ alkenyl,C₄-C₁₀ alkynyl, or C₄-C₁₀ acyl. In other instances, R⁴ is an optionallysubstituted C₁₂-C₂₀ or C₁₄-C₂₂ alkyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkenyl,C₁₂-C₂₀ or C₁₄-C₂₂ alkynyl, or C₁₂-C₂₀ or C₁₄-C₂₂ acyl, and R⁵ is anoptionally substituted C₄-C₈ or C₆ alkyl, C₄-C₈ or C₆ alkenyl, C₄-C₈ orC₆ alkynyl, or C₄-C₈ or C₆ acyl. In certain instances, R⁴ is anoptionally substituted C₄-C₁₀ alkyl, C₄-C₁₀ alkenyl, C₄-C₁₀ alkynyl, orC₄-C₁₀ acyl, and R⁵ is an optionally substituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄alkenyl, C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl. In certain other instances,R⁴ is an optionally substituted C₄-C₈ or C₆ alkyl, C₄-C₈ or C₆ alkenyl,C₄-C₈ or C₆ alkynyl, or C₄-C₈ or C₆ acyl, and R⁵ is an optionallysubstituted C₁₂-C₂₀ or C₁₄-C₂₂ alkyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkenyl,C₁₂-C₂₀ or C₁₄-C₂₂ alkynyl, or C₁₂-C₂₀ or C₁₄-C₂₂ acyl. In particularembodiments, one or more of the optionally substituted cyclic alkylgroups present in R⁴ and/or R⁵ are as described above.

In some groups of embodiments to the cationic lipid of Formula II, R⁴and R⁵ are either the same or different and are independently selectedfrom the group consisting of:

In other groups of embodiments to the cationic lipid of Formula II, oneof R⁴ or R⁵ is selected from the group consisting of:

and the other of R⁴ or R⁵ is selected from the group consisting of:

In certain embodiments, Z is an optionally substituted C₁-C₂ alkyl,C₁-C₃ alkyl, C₁-C₄ alkyl, C₁-C₅ alkyl, C₂-C₃ alkyl, C₂-C₄ alkyl, C₂-C₅alkyl, C₂-C₆ alkyl, C₃-C₄ alkyl, C₃-C₅ alkyl, C₃-C₆ alkyl, C₄-C₅ alkyl,C₄-C₆ alkyl, C₅-C₆ alkyl, C₂-C₃ alkenyl, C₂-C₄ alkenyl, C₂-C₅ alkenyl,C₂-C₆ alkenyl, C₃-C₄ alkenyl, C₃-C₅ alkenyl, C₃-C₆ alkenyl, C₄-C₅alkenyl, C₄-C₆ alkenyl, C₅-C₆ alkenyl, C₂-C₃ alkynyl, C₂-C₄ alkynyl,C₂-C₅ alkynyl, C₂-C₆ alkynyl, C₃-C₄ alkynyl, C₃-C₅ alkynyl, C₃-C₆alkynyl, C₄-C₅ alkynyl, C₄-C₆ alkynyl, or C₅-C₆ alkynyl. In oneparticular embodiment, Z is (CH₂)_(q) and q is 0, 1, 2, 3, 4, 5, or 6(e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 2-3, 2-4, 2-5, or 2-6). In a preferredembodiment, q is 2. In certain other embodiments, q is 1 or 3.

In particular embodiments, the cationic lipid of Formula II has thefollowing structure:

or salts thereof, wherein R¹, R², R³, R⁴, R⁵, X, Y, m, n, p, and q arethe same as described above.

In some embodiments, the cationic lipid of Formula II forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula II is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

In particularly preferred embodiments, the cationic lipid of Formula IIhas one of the following structures:

In yet another aspect, the present invention provides a cationic lipidof Formula III having the following structure:

or salts thereof, wherein:

-   -   R¹ and R² are either the same or different and are independently        hydrogen (H) or an optionally substituted C₁-C₆ alkyl, C₂-C₆        alkenyl, or C₂-C₆ alkynyl, or R¹ and R² may join to form an        optionally substituted heterocyclic ring;    -   R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to        provide a quaternary amine;    -   R⁴ and R⁵ are either the same or different and are independently        an optionally substituted C₁₀-C₂₄ alkyl, C₁₀-C₂₄ alkenyl,        C₁₀-C₂₄ alkynyl, or C₁₀-C₂₄ acyl, wherein at least one of R⁴ and        R⁵ comprises at least 1, 2, 3, 4, 5, 6, or more optionally        substituted cyclic alkyl groups (e.g., 1-2, 1-3, 1-4, 1-5, 1-6,        2-3, 2-4, 2-5, or 2-6 optionally substituted cyclic alkyl        groups);    -   X is O, S, N(R⁶), C(O), C(O)O, OC(O), C(O)N(R⁶), N(R⁶)C(O),        OC(O)N(R⁶), N(R⁶)C(O)O, C(O)S, C(S)O, S(O), S(O)(O), C(S), or an        optionally substituted heterocyclic ring, wherein R⁶ is        hydrogen (H) or an optionally substituted C₁-C₁₀ alkyl, C₂-C₁₀        alkenyl, or C₂-C₁₀ alkynyl; and    -   Y is either absent or is an optionally substituted C₁-C₆ alkyl,        C₂-C₆ alkenyl, or C₂-C₆ alkynyl.

In some embodiments, R¹ and R² are each independently hydrogen (H) or anoptionally substituted C₁-C₂ alkyl, C₁-C₃ alkyl, C₁-C₄ alkyl, C₁-C₅alkyl, C₂-C₃ alkyl, C₂-C₄ alkyl, C₂-C₅ alkyl, C₂-C₆ alkyl, C₃-C₄ alkyl,C₃-C₅ alkyl, C₃-C₆ alkyl, C₄-C₅ alkyl, C₄-C₆ alkyl, C₅-C₆ alkyl, C₂-C₃alkenyl, C₂-C₄ alkenyl, C₂-C₅ alkenyl, C₂-C₆ alkenyl, C₃-C₄ alkenyl,C₃-C₅ alkenyl, C₃-C₆ alkenyl, C₄-C₅ alkenyl, C₄-C₆ alkenyl, C₅-C₆alkenyl, C₂-C₃ alkynyl, C₂-C₄ alkynyl, C₂-C₅ alkynyl, C₂-C₆ alkynyl,C₃-C₄ alkynyl, C₃-C₅ alkynyl, C₃-C₆ alkynyl, C₄-C₅ alkynyl, C₄-C₆alkynyl, or C₅-C₆ alkynyl. In particular embodiments, R¹ and R² are bothmethyl groups, both ethyl groups, or a combination of one methyl groupand one ethyl group. In certain instances, R³ is absent when the pH isabove the pK_(a) of the cationic lipid and R³ is hydrogen (H) when thepH is below the pK_(a) of the cationic lipid such that the amino headgroup is protonated. In certain other instances, R³ is an optionallysubstituted C₁-C₂ alkyl, C₁-C₃ alkyl, C₁-C₄ alkyl, C₁-C₅ alkyl, C₂-C₃alkyl, C₂-C₄ alkyl, C₂-C₅ alkyl, C₂-C₆ alkyl, C₃-C₄ alkyl, C₃-C₅ alkyl,C₃-C₆ alkyl, C₄-C₅ alkyl, C₄-C₆ alkyl, or C₅-C₆ alkyl to provide aquaternary amine.

In certain embodiments, R¹ and R² are joined to form an optionallysubstituted heterocyclic ring comprising 1, 2, 3, 4, 5, 6, or morecarbon atoms and 1, 2, 3, 4, or more heteroatoms such as nitrogen (N),oxygen (O), sulfur (S), and mixtures thereof. In some embodiments, theoptionally substituted heterocyclic ring comprises from 2 to 5 carbonatoms and from 1 to 3 heteroatoms such as nitrogen (N), oxygen (O),and/or sulfur (S). In certain embodiments, the heterocyclic ringcomprises an optionally substituted imidazole, triazole (e.g.,1,2,3-triazole, 1,2,4-triazole), pyrazole, thiazole, pyrrole, furan,oxazole, isoxazole, oxazoline, oxazolidine, oxadiazole, tetrazole, andthe like. In some instances, the optionally substituted heterocyclicring comprises 5 carbon atoms and 1 nitrogen atom, wherein theheterocyclic ring can be substituted with a substituent such as ahydroxyl (—OH) group at the ortho, meta, and/or para positions. Incertain instances, the heterocyclic ring comprises an optionallysubstituted imidazole group.

In other embodiments, R⁶ is hydrogen (H) or an optionally substitutedC₁-C₂ alkyl, C₁-C₃ alkyl, C₁-C₄ alkyl, C₁-C₅ alkyl, C₁-C₆ alkyl, C₁-C₇alkyl, C₁-C₈ alkyl, C₁-C₉ alkyl, C₂-C₃ alkyl, C₂-C₄ alkyl, C₂-C₅ alkyl,C₂-C₆ alkyl, C₂-C₇ alkyl, C₂-C₈ alkyl, C₂-C₉ alkyl, C₂-C₁₀ alkyl, C₃-C₄alkyl, C₃-C₅ alkyl, C₃-C₆ alkyl, C₃-C₇ alkyl, C₃-C₈ alkyl, C₃-C₉ alkyl,C₃-C₁₀ alkyl, C₄-C₅ alkyl, C₄-C₆ alkyl, C₄-C₇ alkyl, C₄-C₈ alkyl, C₄-C₉alkyl, C₄-C₁₀ alkyl, C₅-C₆ alkyl, C₅-C₇ alkyl, C₅-C₈ alkyl, C₅-C₉ alkyl,C₅-C₁₀ alkyl, C₆-C₇ alkyl, C₆-C₈ alkyl, C₆-C₉ alkyl, C₆-C₁₀ alkyl, C₇-C₈alkyl, C₇-C₉ alkyl, C₇-C₁₀ alkyl, C₈-C₉ alkyl, C₈-C₁₀ alkyl, C₉-C₁₀alkyl, C₂-C₃ alkenyl, C₂-C₄ alkenyl, C₂-C₅ alkenyl, C₂-C₆ alkenyl, C₂-C₇alkenyl, C₂-C₈ alkenyl, C₂-C₉ alkenyl, C₃-C₄ alkenyl, C₃-C₅ alkenyl,C₃-C₆ alkenyl, C₃-C₇ alkenyl, C₃-C₈ alkenyl, C₃-C₉ alkenyl, C₃-C₁₀alkenyl, C₄-C₅ alkenyl, C₄-C₆ alkenyl, C₄-C₇ alkenyl, C₄-C₈ alkenyl,C₄-C₉ alkenyl, C₄-C₁₀ alkenyl, C₅-C₆ alkenyl, C₅-C₇ alkenyl, C₅-C₈alkenyl, C₅-C₉ alkenyl, C₅-C₁₀ alkenyl, C₆-C₇ alkenyl, C₆-C₈ alkenyl,C₆-C₉ alkenyl, C₆-C₁₀ alkenyl, C₇-C₈ alkenyl, C₇-C₉ alkenyl, C₇-C₁₀alkenyl, C₈-C₉ alkenyl, C₈-C₁₀ alkenyl, C₉-C₁₀ alkenyl, C₂-C₃ alkynyl,C₂-C₄ alkynyl, C₂-C₅ alkynyl, C₂-C₆ alkynyl, C₂-C₇ alkynyl, C₂-C₈alkynyl, C₂-C₉ alkynyl, C₃-C₄ alkynyl, C₃-C₅ alkynyl, C₃-C₆ alkynyl,C₃-C₇ alkynyl, C₃-C₈ alkynyl, C₃-C₉ alkynyl, C₃-C₁₀ alkynyl, C₄-C₅alkynyl, C₄-C₆ alkynyl, C₄-C₇ alkynyl, C₄-C₈ alkynyl, C₄-C₉ alkynyl,C₄-C₁₀ alkynyl, C₅-C₆ alkynyl, C₅-C₇ alkynyl, C₅-C₈ alkynyl, C₅-C₉alkynyl, C₅-C₁₀ alkynyl, C₆-C₇ alkynyl, C₆-C₈ alkynyl, C₆-C₉ alkynyl,C₆-C₁₀ alkynyl, C₇-C₈ alkynyl, C₇-C₉ alkynyl, C₇-C₁₀ alkynyl, C₈-C₉alkynyl, C₈-C₁₀ alkynyl, or C₉-C₁₀ alkynyl. In one particularembodiment, R⁶ is selected from the group consisting of hydrogen (H), aC₁ alkyl (methyl) group, and a C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, and C₁₀alkyl, alkenyl, and alkynyl group.

In one embodiment, X is O, C(O)O, C(O)N(R⁶), N(R⁶)C(O)O, or C(O)S. Inone particular embodiment, X is C(O)O. In another particular embodiment,X is N(R⁶)C(O)O and R⁶ is hydrogen (H), a methyl group, or a C₂, C₃, C₄,C₅, C₆, C₇, C₈, C₉, or C₁₀ alkyl, alkenyl, or alkynyl group. In certainother embodiments, X is an optionally substituted heterocyclic ring. Inparticular embodiments, the heterocyclic ring comprises 1, 2, 3, 4, 5,6, or more carbon atoms and 1, 2, 3, 4, or more heteroatoms such asnitrogen (N), oxygen (O), sulfur (S), and mixtures thereof. In someembodiments, the optionally substituted heterocyclic ring comprises from2 to 5 carbon atoms and from 1 to 3 heteroatoms such as nitrogen (N),oxygen (O), and/or sulfur (S). In certain embodiments, the heterocyclicring comprises an optionally substituted imidazole, triazole (e.g.,1,2,3-triazole, 1,2,4-triazole), pyrazole, thiazole, pyrrole, furan,oxazole, isoxazole, oxazoline, oxazolidine, oxadiazole, tetrazole, andthe like.

In certain embodiments, R⁴ and R⁵ are independently an optionallysubstituted C₁₂-C₂₄, C₁₂-C₂₂, C₁₂-C₂₀, C₁₄-C₂₄, C₁₄-C₂₂, C₁₄-C₂₀,C₁₆-C₂₄, C₁₆-C₂₂, or C₁₆-C₂₀ alkyl, alkenyl, alkynyl, or acyl group(i.e., a C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, orC₂₄ alkyl, alkenyl, alkynyl, or acyl group), wherein at least one ormore of (e.g., both) R⁴ and R⁵ independently comprises at least 1, 2, 3,4, 5, or 6 optionally substituted cyclic alkyl groups (e.g., 1-2, 1-3,1-4, 1-5, 1-6, 2-3, 2-4, 2-5, or 2-6 optionally substituted cyclic alkylgroups). In particular embodiments, the at least 1, 2, 3, 4, 5, or 6optionally substituted cyclic alkyl groups present in R⁴ and/or R⁵ areindependently selected from the group consisting of an optionallysubstituted saturated cyclic alkyl group, an optionally substitutedunsaturated cyclic alkyl group, and combinations thereof. In certaininstances, the optionally substituted saturated cyclic alkyl groupcomprises an optionally substituted C₃₋₈ cycloalkyl group (e.g.,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, etc.). In preferred embodiments, the optionally substitutedsaturated cyclic alkyl group comprises a cyclopropyl group, optionallycontaining one or more substituents and/or heteroatoms. In otherinstances, the optionally substituted unsaturated cyclic alkyl groupcomprises an optionally substituted C₃₋₈ cycloalkenyl group (e.g.,cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl,cyclooctenyl, etc.). In particular embodiments, R⁴ and R⁵ are the samelength (e.g., C₁₂-C₂₀ or C₁₈ alkyl chains) and contain the same numberand type of cyclic alkyl group (e.g., one, two, or three C₃₋₈ cycloalkylgroups such as one, two, or three cyclopropyl groups on each of R⁴ andR⁵).

In certain other embodiments, one of R⁴ or R⁵ comprises at least 1, 2,3, 4, 5, or 6 sites of unsaturation (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 2-3,2-4, 2-5, or 2-6 sites of unsaturation) or a substituted alkyl or acylgroup, and the other side-chain comprises at least 1, 2, 3, 4, 5, or 6optionally substituted cyclic alkyl groups (e.g., 1-2, 1-3, 1-4, 1-5,1-6, 2-3, 2-4, 2-5, or 2-6 optionally substituted cyclic alkyl groups).In embodiments where one of R⁴ or R⁵ comprises at least 1, 2, 3, 4, 5,or 6 sites of unsaturation, the unsaturated side-chain may comprise adodecenyl moiety, a tetradecenyl (e.g., myristoleyl) moiety, ahexadecenyl (e.g., palmitoleyl) moiety, an octadecenyl (e.g., oleyl)moiety, an icosenyl moiety, a dodecadienyl moiety, a tetradecadienylmoiety, a hexadecadienyl moiety, an octadecadienyl moiety, anicosadienyl moiety, a dodecatrienyl moiety, a tetradectrienyl moiety, ahexadecatrienyl moiety, an octadecatrienyl moiety, an icosatrienylmoiety, or an acyl derivative thereof (e.g., oleoyl, linoleoyl,linolenoyl, γ-linolenoyl, etc.). In some instances, the octadecadienylmoiety is a linoleyl moiety. In other instances, the octadecatrienylmoiety is a linolenyl moiety or a γ-linolenyl moiety. In embodimentswhere one of R⁴ or R⁵ comprises a branched alkyl or acyl group (e.g., asubstituted alkyl or acyl group), the branched alkyl or acyl group maycomprise a C₁₂-C₂₄ alkyl or acyl having at least 1-6 (e.g., 1, 2, 3, 4,5, 6, or more) C₁-C₆ alkyl substituents. In particular embodiments, thebranched alkyl or acyl group comprises a C₁₂-C₂₀ or C₁₄-C₂₂ alkyl oracyl with 1-6 (e.g., 1, 2, 3, 4, 5, 6) C₁-C₄ alkyl (e.g., methyl, ethyl,propyl, or butyl) substituents. In some embodiments, the branched alkylgroup comprises a phytanyl (3,7,11,15-tetramethyl-hexadecanyl) moietyand the branched acyl group comprises a phytanoyl(3,7,11,15-tetramethyl-hexadecanoyl) moiety.

In particular embodiments, R⁴ and R⁵ are both independently selectedC₁₂-C₂₀ alkyl groups (i.e., C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, orC₂₀ alkyl groups) having at least 1, 2, 3, 4, 5, or 6 optionallysubstituted cyclic alkyl groups (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 2-3,2-4, 2-5, or 2-6 optionally substituted cyclic alkyl groups). Inpreferred embodiments, R⁴ and R⁵ are both C₁₈ alkyl groups having atleast one, two, three, or more optionally substituted cyclic alkylgroups such as, for example, an optionally substituted C₃₋₈ cycloalkylgroup (e.g., a cyclopropyl group, optionally containing one or moresubstituents and/or heteroatoms). In certain embodiments, each of theoptionally substituted cyclic alkyl groups is independently selected andcan be the same cyclic alkyl group (e.g., all cyclopropyl groups) ordifferent cyclic alkyl groups (e.g., cyclopropyl and other cycloalkyl,heterocycloalkyl, cycloalkenyl, and/or heterocycloalkenyl groups).

In preferred embodiments, the optionally substituted cyclic alkyl groupspresent in R⁴ and/or R⁵ are located at the site(s) of unsaturation inthe corresponding unsaturated side-chain. As a non-limiting example, oneor both of R⁴ and R⁵ are C₁₈ alkyl groups having 1, 2, or 3 optionallysubstituted cyclic alkyl groups, wherein the optionally substitutedcyclic alkyl groups (e.g., independently selected cyclopropyl groups)are located at one or more (e.g., all) of the sites of unsaturationpresent in a corresponding linoleyl moiety, linolenyl moiety, orγ-linolenyl moiety.

In alternative embodiments to the cationic lipid of Formula III, R⁴ andR⁵ are different and are independently an optionally substituted C₁-C₂₄alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl, or C₁-C₂₄ acyl, wherein at leastone of R⁴ and R⁵ comprises at least one optionally substituted cyclicalkyl group. In certain embodiments, R⁴ and R⁵ are different and areindependently an optionally substituted C₄-C₂₀ alkyl, C₄-C₂₀ alkenyl,C₄-C₂₀ alkynyl, or C₄-C₂₀ acyl. In some instances, R⁴ is an optionallysubstituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄ alkenyl, C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄acyl, and R⁵ is an optionally substituted C₄-C₁₀ alkyl, C₄-C₁₀ alkenyl,C₄-C₁₀ alkynyl, or C₄-C₁₀ acyl. In other instances, R⁴ is an optionallysubstituted C₁₂-C₂₀ or C₁₄-C₂₂ alkyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkenyl,C₁₂-C₂₀ or C₁₄-C₂₂ alkynyl, or C₁₂-C₂₀ or C₁₄-C₂₂ acyl, and R⁵ is anoptionally substituted C₄-C₈ or C₆ alkyl, C₄-C₈ or C₆ alkenyl, C₄-C₈ orC₆ alkynyl, or C₄-C₈ or C₆ acyl. In certain instances, R⁴ is anoptionally substituted C₄-C₁₀ alkyl, C₄-C₁₀ alkenyl, C₄-C₁₀ alkynyl, orC₄-C₁₀ acyl, and R⁵ is an optionally substituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄alkenyl, C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl. In certain other instances,R⁴ is an optionally substituted C₄-C₈ or C₆ alkyl, C₄-C₈ or C₆ alkenyl,C₄-C₈ or C₆ alkynyl, or C₄-C₈ or C₆ acyl, and R⁵ is an optionallysubstituted C₁₂-C₂₀ or C₁₄-C₂₂ alkyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkenyl,C₁₂-C₂₀ or C₁₄-C₂₂ alkynyl, or C₁₂-C₂₀ or C₁₄-C₂₂ acyl. In particularembodiments, one or more of the optionally substituted cyclic alkylgroups present in R⁴ and/or R⁵ are as described above.

In some groups of embodiments to the cationic lipid of Formula III, R⁴and R⁵ are either the same or different and are independently selectedfrom the group consisting of:

In other groups of embodiments to the cationic lipid of Formula III, oneof R⁴ or R⁵ is selected from the group consisting of:

and the other of R⁴ or R⁵ is selected from the group consisting of:

In certain embodiments, Y is an optionally substituted C₁-C₂ alkyl,C₁-C₃ alkyl, C₁-C₄ alkyl, C₁-C₅ alkyl, C₂-C₃ alkyl, C₂-C₄ alkyl, C₂-C₅alkyl, C₂-C₆ alkyl, C₃-C₄ alkyl, C₃-C₅ alkyl, C₃-C₆ alkyl, C₄-C₅ alkyl,C₄-C₆ alkyl, C₅-C₆ alkyl, C₂-C₃ alkenyl, C₂-C₄ alkenyl, C₂-C₅ alkenyl,C₂-C₆ alkenyl, C₃-C₄ alkenyl, C₃-C₅ alkenyl, C₃-C₆ alkenyl, C₄-C₅alkenyl, C₄-C₆ alkenyl, C₅-C₆ alkenyl, C₂-C₃ alkynyl, C₂-C₄ alkynyl,C₂-C₅ alkynyl, C₂-C₆ alkynyl, C₃-C₄ alkynyl, C₃-C₅ alkynyl, C₃-C₆alkynyl, C₄-C₅ alkynyl, C₄-C₆ alkynyl, or C₅-C₆ alkynyl. In oneparticular embodiment, Y is (CH₂)_(n) and n is 0, 1, 2, 3, 4, 5, or 6(e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 2-3, 2-4, 2-5, 2-6, 3-4, 3-5, or 3-6).In a preferred embodiment, n is 3. In certain other embodiments, n is 2or 4.

In particular embodiments, the cationic lipid of Formula III has thefollowing structure:

or salts thereof, wherein R¹, R², R³, R⁴, R⁵, X, and n are the same asdescribed above.

In some embodiments, the cationic lipid of Formula III forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula III is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

In particularly preferred embodiments, the cationic lipid of Formula IIIhas one of the following structures:

The compounds of the invention may be prepared by known organicsynthesis techniques, including the methods described in the Examples.In some embodiments, the synthesis of the cationic lipids of theinvention may require the use of protecting groups. Protecting groupmethodology is well known to those skilled in the art (see, e.g.,PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, Green, T. W. et. al.,Wiley-Interscience, New York City, 1999). Briefly, protecting groupswithin the context of this invention are any group that reduces oreliminates the unwanted reactivity of a functional group. A protectinggroup can be added to a functional group to mask its reactivity duringcertain reactions and then removed to reveal the original functionalgroup. In certain instances, an “alcohol protecting group” is used. An“alcohol protecting group” is any group which decreases or eliminatesthe unwanted reactivity of an alcohol functional group. Protectinggroups can be added and removed using techniques well known in the art.

In certain embodiments, the cationic lipids of the present inventionhave at least one protonatable or deprotonatable group, such that thelipid is positively charged at a pH at or below physiological pH (e.g.,pH 7.4), and neutral at a second pH, preferably at or abovephysiological pH. It will be understood by one of ordinary skill in theart that the addition or removal of protons as a function of pH is anequilibrium process, and that the reference to a charged or a neutrallipid refers to the nature of the predominant species and does notrequire that all of the lipid be present in the charged or neutral form.Lipids that have more than one protonatable or deprotonatable group, orwhich are zwiterrionic, are not excluded from use in the invention.

In certain other embodiments, protonatable lipids according to theinvention have a pK_(a) of the protonatable group in the range of about4 to about 11. Most preferred is a pK_(a) of about 4 to about 7, becausethese lipids will be cationic at a lower pH formulation stage, whileparticles will be largely (though not completely) surface neutralized atphysiological pH of around pH 7.4. One of the benefits of this pK_(a) isthat at least some nucleic acid associated with the outside surface ofthe particle will lose its electrostatic interaction at physiological pHand be removed by simple dialysis, thus greatly reducing the particle'ssusceptibility to clearance.

IV. Active Agents

Active agents (e.g., therapeutic agents) include any molecule orcompound capable of exerting a desired effect on a cell, tissue, tumor,organ, or subject. Such effects may be, e.g., biological, physiological,and/or cosmetic. Active agents may be any type of molecule or compoundincluding, but not limited to, nucleic acids, peptides, polypeptides,small molecules, and mixtures thereof. Non-limiting examples of nucleicacids include interfering RNA molecules (e.g., dsRNA such as siRNA,Dicer-substrate dsRNA, shRNA, aiRNA, and/or miRNA), antisenseoligonucleotides, plasmids, ribozymes, immunostimulatoryoligonucleotides, and mixtures thereof. Examples of peptides orpolypeptides include, without limitation, antibodies (e.g., polyclonalantibodies, monoclonal antibodies, antibody fragments; humanizedantibodies, recombinant antibodies, recombinant human antibodies, and/orPrimatized™ antibodies), cytokines, growth factors, apoptotic factors,differentiation-inducing factors, cell-surface receptors and theirligands, hormones, and mixtures thereof. Examples of small moleculesinclude, but are not limited to, small organic molecules or compoundssuch as any conventional agent or drug known to those of skill in theart.

In some embodiments, the active agent is a therapeutic agent, or a saltor derivative thereof. Therapeutic agent derivatives may betherapeutically active themselves or they may be prodrugs, which becomeactive upon further modification. Thus, in one embodiment, a therapeuticagent derivative retains some or all of the therapeutic activity ascompared to the unmodified agent, while in another embodiment, atherapeutic agent derivative is a prodrug that lacks therapeuticactivity, but becomes active upon further modification.

In preferred embodiments, the lipid particles described herein areassociated with a nucleic acid, resulting in a nucleic acid-lipidparticle (e.g., SNALP). Non-limiting exemplary embodiments related toselecting, synthesizing, and modifying nucleic acids such as siRNA,Dicer-substrate dsRNA, shRNA, aiRNA, miRNA, antisense oligonucleotides,ribozymes, and immunostimulatory oligonucleotides are described, forexample, in U.S. Patent Publication No. 20070135372; in U.S. PatentPublication No. 20110076335; and in PCT Publication No. WO 2010/105372,the disclosures of which are each herein incorporated by reference intheir entirety for all purposes.

In certain embodiments, the nucleic acid (e.g., interfering RNA)component of the nucleic acid-lipid particle (e.g., SNALP) comprises atleast one modified nucleotide (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, or more modified nucleotides). In certain instances, thenucleic acid (e.g., interfering RNA such as an siRNA) comprises modifiednucleotides including, but not limited to, 2′-O-methyl (2′OMe)nucleotides, 2′-deoxy-2′-fluoro (2′F) nucleotides, 2′-deoxy nucleotides,2′-O-(2-methoxyethyl) (MOE) nucleotides, locked nucleic acid (LNA)nucleotides, 5-C-methyl nucleotides, 4′-thio nucleotides, 2′-aminonucleotides, 2′-C-allyl nucleotides, and mixtures thereof. In particularembodiments, the modified interfering RNA (e.g., modified siRNA) isgenerally less immunostimulatory than a corresponding unmodifiedinterfering RNA (e.g., unmodified siRNA) sequence and retains RNAiactivity against the target gene of interest. In some embodiments, themodified interfering RNA (e.g., modified siRNA) contains at least one2′OMe purine or pyrimidine nucleotide such as a 2′OMe-guanosine,2′OMe-uridine, 2′OMe-adenosine, and/or 2′OMe-cytosine nucleotide. Themodified nucleotides can be present in one strand (i.e., sense orantisense) or both strands of the interfering RNA (e.g., siRNA). In somepreferred embodiments, one or more of the uridine and/or guanosinenucleotides are modified (e.g., 2′OMe-modified) in one strand (i.e.,sense or antisense) or both strands of the interfering RNA (e.g.,siRNA). In these embodiments, the modified interfering RNA (e.g.,modified siRNA) can further comprise one or more modified (e.g.,2′OMe-modified) adenosine and/or modified (e.g., 2′OMe-modified)cytosine nucleotides. In other preferred embodiments, only uridineand/or guanosine nucleotides are modified (e.g., 2′OMe-modified) in onestrand (i.e., sense or antisense) or both strands of the interfering RNA(e.g., siRNA). The interfering RNA (e.g., siRNA) sequences may haveoverhangs (e.g., 3′ or 5′ overhangs as described in Elbashir et al.,Genes Dev., 15:188 (2001) or Nykinen et al., Cell, 107:309 (2001)), ormay lack overhangs (i.e., have blunt ends). The interfering RNA (e.g.,siRNA) sequences may comprise one or more modified nucleotides in thedouble-stranded (duplex) region and/or in one or both of the overhangs(e.g., 3′ overhangs) when present.

The nucleic acid (e.g., interfering RNA) component of the nucleicacid-lipid particle (e.g., SNALP) can be used to downregulate or silencethe translation (i.e., expression) of a gene of interest. Non-limitingexamples of genes of interest include genes associated with metabolicdiseases and disorders (e.g., liver diseases and disorders), genesassociated with cell proliferation, tumorigenesis, and/or celltransformation (e.g., a cell proliferative disorder such as cancer),angiogenic genes, receptor ligand genes, immunomodulator genes (e.g.,those associated with inflammatory and autoimmune responses), genesassociated with viral infection and survival, and genes associated withneurodegenerative disorders. See, e.g., U.S. Patent Publication No.20110076335 for a description of exemplary target genes (including theirGenbank Accession Nos.) which may be downregulated or silenced by thenucleic acid (e.g., interfering RNA) of the nucleic acid-lipid particle(e.g., SNALP).

Non-limiting examples of gene sequences associated with tumorigenesis orcell transformation include polo-like kinase 1 (PLK-1), cyclin-dependentkinase 4 (CDK4), COP1, ring-box 1 (RBX1), WEE1, Eg5 (KSP, KIF11),forkhead box M1 (FOXM1), RAM2 (R1, CDCA7L), XIAP, CSN5 (JAB1), andHDAC2. Non-limiting examples of gene sequences associated with metabolicdiseases and disorders include apolipoprotein B (APOB), apolipoproteinCIII (APOC3), apolipoprotein E (APOE), proprotein convertasesubtilisin/kexin type 9 (PCSK9), diacylglycerol O-acyltransferase type 1(DGAT1), and diacylglyerol O-acyltransferase type 2 (DGAT2).Non-limiting examples of gene sequences associated with viral infectionand survival include host factors such as tissue factor (TF) or nucleicacid sequences from Filoviruses such as Ebola virus and Marburg virus(e.g., VP30, VP35, nucleoprotein (NP), polymerase protein (L-pol), VP40,glycoprotein (GP), and VP24); Arenaviruses such as Lassa virus (e.g.,NP, GP, L, and/or Z genes), Junin virus, Machupo virus, Guanarito virus,and Sabia virus; Hepatitis viruses such as Hepatitis A, B, C, D, and Eviruses; Influenza viruses such as Influenza A, B, and C viruses; HumanImmunodeficiency Virus (HIV); Herpes viruses; and Human PapillomaViruses (HPV).

In other embodiments, the active agent associated with the lipidparticles of the invention may comprise one or more therapeuticproteins, polypeptides, or small organic molecules or compounds.Non-limiting examples of such therapeutically effective agents or drugsinclude oncology drugs (e.g., chemotherapy drugs, hormonal therapeuticagents, immunotherapeutic agents, radiotherapeutic agents, etc.),lipid-lowering agents, anti-viral drugs, anti-inflammatory compounds,antidepressants, stimulants, analgesics, antibiotics, birth controlmedication, antipyretics, vasodilators, anti-angiogenics, cytovascularagents, signal transduction inhibitors, cardiovascular drugs such asanti-arrhythmic agents, hormones, vasoconstrictors, and steroids. Theseactive agents may be administered alone in the lipid particles of theinvention, or in combination (e.g., co-administered) with lipidparticles of the invention comprising nucleic acid such as interferingRNA. Non-limiting examples of these types of active agents aredescribed, e.g., in U.S. Patent Publication No. 20110076335, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes.

V. Lipid Particles

In certain aspects, the present invention provides lipid particlescomprising one or more of the cationic (amino) lipids or salts thereofdescribed herein. In some embodiments, the lipid particles of theinvention further comprise one or more non-cationic lipids. In otherembodiments, the lipid particles further comprise one or more conjugatedlipids capable of reducing or inhibiting particle aggregation. Inadditional embodiments, the lipid particles further comprise one or moreactive agents or therapeutic agents such as therapeutic nucleic acids(e.g., interfering RNA such as siRNA).

Lipid particles include, but are not limited to, lipid vesicles such asliposomes. As used herein, a lipid vesicle includes a structure havinglipid-containing membranes enclosing an aqueous interior. In particularembodiments, lipid vesicles comprising one or more of the cationiclipids described herein are used to encapsulate nucleic acids within thelipid vesicles. In other embodiments, lipid vesicles comprising one ormore of the cationic lipids described herein are complexed with nucleicacids to form lipoplexes.

The lipid particles of the invention typically comprise an active agentor therapeutic agent, a cationic lipid, a non-cationic lipid, and aconjugated lipid that inhibits aggregation of particles. In someembodiments, the active agent or therapeutic agent is fully encapsulatedwithin the lipid portion of the lipid particle such that the activeagent or therapeutic agent in the lipid particle is resistant in aqueoussolution to enzymatic degradation, e.g., by a nuclease or protease. Inother embodiments, the lipid particles described herein aresubstantially non-toxic to mammals such as humans. The lipid particlesof the invention typically have a mean diameter of from about 30 nm toabout 150 nm, from about 40 nm to about 150 nm, from about 50 nm toabout 150 nm, from about 60 nm to about 130 nm, from about 70 nm toabout 110 nm, or from about 70 to about 90 nm. The lipid particles ofthe invention also typically have a lipid:therapeutic agent (e.g.,lipid:nucleic acid) ratio (mass/mass ratio) of from about 1:1 to about100:1, from about 1:1 to about 50:1, from about 2:1 to about 25:1, fromabout 3:1 to about 20:1, from about 5:1 to about 15:1, or from about 5:1to about 10:1.

In preferred embodiments, the lipid particles of the invention areserum-stable nucleic acid-lipid particles (SNALP) which comprise aninterfering RNA (e.g., dsRNA such as siRNA, Dicer-substrate dsRNA,shRNA, aiRNA, and/or miRNA), a cationic lipid (e.g., one or morecationic lipids of Formulas I-III or salts thereof as set forth herein),a non-cationic lipid (e.g., mixtures of one or more phospholipids andcholesterol), and a conjugated lipid that inhibits aggregation of theparticles (e.g., one or more PEG-lipid conjugates). The SNALP maycomprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more unmodifiedand/or modified interfering RNA molecules (e.g., siRNA). Nucleicacid-lipid particles and their method of preparation are described in,e.g., U.S. Pat. Nos. 5,753,613; 5,785,992; 5,705,385; 5,976,567;5,981,501; 6,110,745; and 6,320,017; and PCT Publication No. WO96/40964, the disclosures of which are each herein incorporated byreference in their entirety for all purposes.

In the nucleic acid-lipid particles of the invention, the nucleic acidmay be fully encapsulated within the lipid portion of the particle,thereby protecting the nucleic acid from nuclease degradation. Inpreferred embodiments, a SNALP comprising a nucleic acid such as aninterfering RNA is fully encapsulated within the lipid portion of theparticle, thereby protecting the nucleic acid from nuclease degradation.In certain instances, the nucleic acid in the SNALP is not substantiallydegraded after exposure of the particle to a nuclease at 37° C. for atleast about 20, 30, 45, or 60 minutes. In certain other instances, thenucleic acid in the SNALP is not substantially degraded after incubationof the particle in serum at 37° C. for at least about 30, 45, or 60minutes or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. In other embodiments, thenucleic acid is complexed with the lipid portion of the particle. One ofthe benefits of the formulations of the present invention is that thenucleic acid-lipid particle compositions are substantially non-toxic tomammals such as humans.

The term “fully encapsulated” indicates that the nucleic acid in thenucleic acid-lipid particle is not significantly degraded after exposureto serum or a nuclease assay that would significantly degrade free DNAor RNA. In a fully encapsulated system, preferably less than about 25%of the nucleic acid in the particle is degraded in a treatment thatwould normally degrade 100% of free nucleic acid, more preferably lessthan about 10%, and most preferably less than about 5% of the nucleicacid in the particle is degraded. “Fully encapsulated” also indicatesthat the nucleic acid-lipid particles are serum-stable, that is, thatthey do not rapidly decompose into their component parts upon in vivoadministration.

In the context of nucleic acids, full encapsulation may be determined byperforming a membrane-impermeable fluorescent dye exclusion assay, whichuses a dye that has enhanced fluorescence when associated with nucleicacid. Specific dyes such as OliGreen® and RiboGreen® (Invitrogen Corp.;Carlsbad, Calif.) are available for the quantitative determination ofplasmid DNA, single-stranded deoxyribonucleotides, and/or single- ordouble-stranded ribonucleotides. Encapsulation is determined by addingthe dye to a liposomal formulation, measuring the resultingfluorescence, and comparing it to the fluorescence observed uponaddition of a small amount of nonionic detergent. Detergent-mediateddisruption of the liposomal bilayer releases the encapsulated nucleicacid, allowing it to interact with the membrane-impermeable dye. Nucleicacid encapsulation may be calculated as E=(I_(o)−I)/I_(o), where I andI_(o) refer to the fluorescence intensities before and after theaddition of detergent (see, Wheeler et al., Gene Ther., 6:271-281(1999)).

In other embodiments, the present invention provides a nucleicacid-lipid particle (e.g., SNALP) composition comprising a plurality ofnucleic acid-lipid particles.

In some instances, the SNALP composition comprises nucleic acid that isfully encapsulated within the lipid portion of the particles, such thatfrom about 30% to about 100%, from about 40% to about 100%, from about50% to about 100%, from about 60% to about 100%, from about 70% to about100%, from about 80% to about 100%, from about 90% to about 100%, fromabout 30% to about 95%, from about 40% to about 95%, from about 50% toabout 95%, from about 60% to about 95%, from about 70% to about 95%,from about 80% to about 95%, from about 85% to about 95%, from about 90%to about 95%, from about 30% to about 90%, from about 40% to about 90%,from about 50% to about 90%, from about 60% to about 90%, from about 70%to about 90%, from about 80% to about 90%, or at least about 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% (or any fraction thereof or rangetherein) of the particles have the nucleic acid encapsulated therein.

In other instances, the SNALP composition comprises nucleic acid that isfully encapsulated within the lipid portion of the particles, such thatfrom about 30% to about 100%, from about 40% to about 100%, from about50% to about 100%, from about 60% to about 100%, from about 70% to about100%, from about 80% to about 100%, from about 90% to about 100%, fromabout 30% to about 95%, from about 40% to about 95%, from about 50% toabout 95%, from about 60% to about 95%, from about 70% to about 95%,from about 80% to about 95%, from about 85% to about 95%, from about 90%to about 95%, from about 30% to about 90%, from about 40% to about 90%,from about 50% to about 90%, from about 60% to about 90%, from about 70%to about 90%, from about 80% to about 90%, or at least about 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% (or any fraction thereof or rangetherein) of the input nucleic acid is encapsulated in the particles.

Depending on the intended use of the lipid particles of the invention,the proportions of the components can be varied and the deliveryefficiency of a particular formulation can be measured using, e.g., anendosomal release parameter (ERP) assay.

In particular embodiments, the present invention provides a lipidparticle (e.g., SNALP) composition comprising a plurality of lipidparticles described herein and an antioxidant. In certain instances, theantioxidant in the lipid particle composition reduces, prevents, and/orinhibits the degradation of a cationic lipid (e.g., a polyunsaturatedcationic lipid) present in the lipid particle. In instances wherein theactive agent is a therapeutic nucleic acid such as an interfering RNA(e.g., siRNA), the antioxidant in the lipid particle compositionreduces, prevents, and/or inhibits the degradation of the nucleic acidpayload, e.g., by reducing, preventing, and/or inhibiting the oxidationof the cationic lipid, by reducing, preventing, and/or inhibiting thedegradation of the nucleic acid payload, by reducing, preventing, and/orinhibiting the desulfurization of a phosphorothioate (PS)-modifiednucleic acid payload, and/or by stabilizing both the lipid and nucleicacid components.

Examples of antioxidants include, but are not limited to, metalchelators (e.g., ethylenediaminetetraacetic acid (EDTA), citrate, andthe like), primary antioxidants (e.g., vitamin E isomers such asα-tocopherol or a salt thereof, butylated hydroxyanisole (BHA),butylhydroxytoluene (BHT), tert-butylhydroquinone (TBHQ), and the like),secondary antioxidants (e.g., ascorbic acid, ascorbyl palmitate,cysteine, glutathione, α-lipoic acid, and the like), salts thereof, andmixtures thereof. If needed, the antioxidant is typically present in anamount sufficient to prevent, inhibit, and/or reduce the degradation ofthe cationic lipid and/or active agent present in the lipid particle. Inparticular embodiments, the antioxidant comprises EDTA or a salt thereof(e.g., from about 20 mM to about 100 mM), alone or in combination with aprimary antioxidant such as α-tocopherol or a salt thereof (e.g., fromabout 0.02 mol % to about 0.5 mol %) and/or secondary antioxidant suchas ascorbyl palmitate or a salt thereof (e.g., from about 0.02 mol % toabout 5.0 mol %). An antioxidant such as EDTA may be included at anystep or at multiple steps in the lipid particle formation processdescribed in Section VI (e.g., prior to, during, and/or after lipidparticle formation).

Additional embodiments related to methods of preventing the degradationof cationic lipids and/or active agents (e.g., therapeutic nucleicacids) present in lipid particles, compositions comprising lipidparticles stabilized by these methods, methods of making these lipidparticles, and methods of delivering and/or administering these lipidparticles are described in PCT Application No. PCT/CA2010/001919, filedDec. 1, 2010, the disclosure of which is herein incorporated byreference in its entirety for all purposes.

In one aspect, the lipid particles of the invention may include atargeting lipid. In some embodiments, the targeting lipid comprises aGalNAc moiety (i.e., an N-galactosamine moiety). As a non-limitingexample, a targeting lipid comprising a GalNAc moiety can include thosedescribed in U.S. application Ser. No. 12/328,669, filed Dec. 4, 2008,the disclosure of which is herein incorporated by reference in itsentirety for all purposes. A targeting lipid can also include any otherlipid (e.g., targeting lipid) known in the art, for example, asdescribed in U.S. application Ser. No. 12/328,669 or PCT Publication No.WO 2008/042973, the contents of each of which are incorporated herein byreference in their entirety for all purposes. In some embodiments, thetargeting lipid includes a plurality of GalNAc moieties, e.g., two orthree GalNAc moieties. In some embodiments, the targeting lipid containsa plurality, e.g., two or three N-acetylgalactosamine (GalNAc) moieties.In some embodiments, the lipid in the targeting lipid is1,2-Di-O-hexadecyl-sn-glyceride (i.e., DSG). In some embodiments, thetargeting lipid includes a PEG moiety (e.g., a PEG moiety having amolecular weight of at least about 500 Da, such as about 1000 Da, 1500Da, 2000 Da or greater), for example, the targeting moiety is connectedto the lipid via a PEG moiety. Examples of GalNAc targeting lipidsinclude, but are not limited to, (GalNAc)₃-PEG-DSG, (GalNAc)₃-PEG-LCO,and mixtures thereof.

In some embodiments, the targeting lipid includes a folate moiety. Forexample, a targeting lipid comprising a folate moiety can include thosedescribed in U.S. application Ser. No. 12/328,669, the disclosure ofwhich is herein incorporated by reference in its entirety for allpurposes. Examples of folate targeting lipids include, but are notlimited to,1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethyleneglycol)-2000] (ammonium salt) (Folate-PEG-DSPE), Folate-PEG2000-DSG,Folate-PEG3400-DSG, and mixtures thereof.

In another aspect, the lipid particles of the invention may furthercomprise one or more apolipoproteins. As used herein, the term“apolipoprotein” or “lipoprotein” refers to apolipoproteins known tothose of skill in the art and variants and fragments thereof and toapolipoprotein agonists, analogues, or fragments thereof described in,e.g., PCT Publication No. WO 2010/0088537, the disclosure of which isherein incorporated by reference in its entirety for all purposes.Suitable apolipoproteins include, but are not limited to, ApoA-I,ApoA-II, ApoA-IV, ApoA-V, and ApoE (e.g., ApoE2, ApoE3, etc.), andactive polymorphic forms, isoforms, variants, and mutants as well asfragments or truncated forms thereof.

Isolated ApoE and/or active fragments and polypeptide analogues thereof,including recombinantly produced forms thereof, are described in U.S.Pat. Nos. 5,672,685; 5,525,472; 5,473,039; 5,182,364; 5,177,189;5,168,045; and 5,116,739, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

A. Cationic Lipids

Any of the novel cationic lipids of Formulas I-III or salts thereof asset forth herein may be used in the lipid particles of the presentinvention (e.g., SNALP), either alone or in combination with one or moreother cationic lipid species or non-cationic lipid species.

Other cationic lipids or salts thereof which may also be included in thelipid particles of the present invention include, but are not limitedto, one or more of the cationic lipids of Formulas I-XXII or saltsthereof as described in U.S. application Ser. No. 13/077,856, filed Mar.31, 2011, one or more of the cationic lipids of Formulas I-XIX or saltsthereof as described in PCT Application No. PCT/CA2010/001919, filedDec. 1, 2010, and/or one or more of the cationic lipids of Formula I orsalts thereof as described in PCT Application No. PCT/GB2011/______,entitled “Novel Cationic Lipids and Methods of Use Thereof,” bearingAttorney Docket No. 86399-010620PC (805952) and/or Reference No.N.114016 PJC/JRN, filed May 12, 2011, the disclosures of which areherein incorporated by reference in their entirety for all purposes.Non-limiting examples of additional suitable cationic lipids include1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA;“XTC2” or “C2K”), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane(DLin-K-DMA), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane(DLin-K-C3-DMA; “C3K”),2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA;“C4K”), 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane(DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane(DLin-K-MPZ), 2,2-dioleoyl-4-dimethylaminomethyl-[1,3]-dioxolane(DO-K-DMA), 2,2-distearoyl-4-dimethylaminomethyl-[1,3]-dioxolane(DS-K-DMA), 2,2-dilinoleyl-4-N-morpholino-[1,3]-dioxolane (DLin-K-MA),2,2-Dilinoleyl-4-trimethylamino-[1,3]-dioxolane chloride(DLin-K-TMA.Cl),2,2-dilinoleyl-4,5-bis(dimethylaminomethyl)-[1,3]-dioxolane(DLin-K²-DMA), 2,2-dilinoleyl-4-methylpiperzine-[1,3]-dioxolane(D-Lin-K-N-methylpiperzine), DLen-C2K-DMA, γ-DLen-C2K-DMA, DPan-C2K-DMA,DPan-C3K-DMA, DLen-C2K-DMA, γ-DLen-C2K-DMA, DPan-C2K-DMA, TLinDMA,C2-TLinDMA, C3-TLinDMA, 1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane(γ-DLenDMA), 1,2-dilinoleyloxy-(N,N-dimethyl)-butyl-4-amine(C2-DLinDMA), 1,2-dilinoleoyloxy-(N,N-dimethyl)-butyl-4-amine(C2-DLinDAP), dilinoleylmethyl-3-dimethylaminopropionate(DLin-M-C2-DMA), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino) butanoate (DLin-M-C3-DMA or “MC3”; also calleddilinoleylmethyl 4-(dimethylamino)butanoate), MC3 Ether(3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-1-amine;also called dilinoleylmethyl 4-(dimethylamino)propyl ether), MC4 Ether(4-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylbutan-1-amine;also called dilinoleylmethyl 4-(dimethylamino)butyl ether), LenMC3,γ-LenMC3, MC3MC, MC2C, MC2MC, MC3 Thioester, MC3 Alkyne, MC3 Amide,Pan-MC3, Pan-MC4, Pan-MC5, analogs thereof, salts thereof, and mixturesthereof.

Examples of yet additional cationic lipids include, but are not limitedto, 1,2-dioeylcarbamoyloxy-3-dimethylaminopropane (DO-C-DAP),1,2-dimyristoleoyl-3-dimethylaminopropane (DMDAP),1,2-dioleoyl-3-trimethylaminopropane chloride (DOTAP.Cl),1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ),3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-dioleylamino)-1,2-propanedio (DOAP),1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(CLinDMA),2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethy-1-(cis,cis-9′,1-2′-octadecadienoxy)propane(CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA),1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP),1,2-N,N′-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP),N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA),N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol),N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE),2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate(DOSPA), dioctadecylamidoglycyl spermine (DOGS), analogs thereof, saltsthereof, and mixtures thereof.

In some embodiments, the additional cationic lipid forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the additional cationic lipid is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

The synthesis of cationic lipids such as DLinDMA and DLenDMA, as well asadditional cationic lipids, is described in U.S. Patent Publication No.20060083780, the disclosure of which is herein incorporated by referencein its entirety for all purposes.

The synthesis of cationic lipids such as DLin-K-C2-DMA, DLin-K-C3-DMA,DLin-K-C4-DMA, DLin-K6-DMA, DLin-K-MPZ, DO-K-DMA, DS-K-DMA, DLin-K-MA,DLin-K-TMA.Cl, DLin-K²-DMA, D-Lin-K-N-methylpiperzine, DLin-M-C2-DMA,DO-C-DAP, DMDAP, and DOTAP.Cl, as well as additional cationic lipids, isdescribed in PCT Publication No. WO 2010/042877, the disclosure of whichis incorporated herein by reference in its entirety for all purposes.

The synthesis of cationic lipids such as DLin-K-DMA, DLin-C-DAP,DLinDAC, DLinMA, DLinDAP, DLin-S-DMA, DLin-2-DMAP, DLinTMA.Cl,DLinTAP.Cl, DLinMPZ, DLinAP, DOAP, and DLin-EG-DMA, as well asadditional cationic lipids, is described in PCT Publication No. WO09/086558, the disclosure of which is herein incorporated by referencein its entirety for all purposes.

The synthesis of cationic lipids such as γ-DLenDMA, DLen-C2K-DMA,γ-DLen-C2K-DMA, DPan-C2K-DMA, DPan-C3K-DMA, DLen-C2K-DMA,γ-DLen-C2K-DMA, DPan-C2K-DMA, TLinDMA, C2-TLinDMA, C3-TLinDMA,C2-DLinDMA, and C2-DLinDAP, as well as additional cationic lipids, isdescribed in PCT Publication No. WO 2011/000106, the disclosure of whichis herein incorporated by reference in its entirety for all purposes.

The synthesis of cationic lipids such as MC3 and MC3 analogs such asLenMC3, γ-LenMC3, MC3MC, MC2C, MC2MC, MC3 Thioester, MC3 Ether, MC4Ether, MC3 Alkyne, MC3 Amide, Pan-MC3, Pan-MC4, and Pan-MC5 is describedin PCT Application No. PCT/GB2011/______, entitled “Novel CationicLipids and Methods of Use Thereof,” bearing Attorney Docket No.86399-010620PC (805952) and/or Reference No. N.11406 PJC/JRN, filed May12, 2011, the disclosure of which is herein incorporated by reference inits entirety for all purposes.

The synthesis of cationic lipids such as CLinDMA, as well as additionalcationic lipids, is described in U.S. Patent Publication No.20060240554, the disclosure of which is herein incorporated by referencein its entirety for all purposes.

The synthesis of a number of other cationic lipids and related analogshas been described in U.S. Pat. Nos. 5,208,036; 5,264,618; 5,279,833;5,283,185; 5,753,613; and 5,785,992; and PCT Publication No. WO96/10390, the disclosures of which are each herein incorporated byreference in their entirety for all purposes. Additionally, a number ofcommercial preparations of cationic lipids can be used, such as, e.g.,LIPOFECTIN® (including DOTMA and DOPE, available from GIBCO/BRL);LIPOFECTAMINE® (including DOSPA and DOPE, available from GIBCO/BRL); andTRANSFECTAM® (including DOGS, available from Promega Corp.).

The synthesis of additional cationic lipids suitable for use in thelipid particles of the present invention is described in PCT PublicationNos. WO 2010/054401, WO 2010/054405, WO 2010/054406, WO 2010/054384, andWO 2010/144740; U.S. Patent Publication No. 20090023673; and U.S.Provisional Application No. 61/287,995, entitled “Methods andCompositions for Delivery of Nucleic Acids,” filed Dec. 18, 2009, thedisclosures of which are herein incorporated by reference in theirentirety for all purposes.

In some embodiments, the cationic lipid comprises from about 45 mol % toabout 90 mol %, from about 45 mol % to about 85 mol %, from about 45 mol% to about 80 mol %, from about 45 mol % to about 75 mol %, from about45 mol % to about 70 mol %, from about 45 mol % to about 65 mol %, fromabout 45 mol % to about 60 mol %, from about 45 mol % to about 55 mol %,from about 50 mol % to about 90 mol %, from about 50 mol % to about 85mol %, from about 50 mol % to about 80 mol %, from about 50 mol % toabout 75 mol %, from about 50 mol % to about 70 mol %, from about 50 mol% to about 65 mol %, from about 50 mol % to about 60 mol %, from about55 mol % to about 65 mol % or from about 55 mol % to about 70 mol % (orany fraction thereof or range therein) of the total lipid present in theparticle.

In certain preferred embodiments, the cationic lipid comprises fromabout 50 mol % to about 58 mol %, from about 51 mol % to about 59 mol %,from about 51 mol % to about 58 mol %, from about 51 mol % to about 57mol %, from about 52 mol % to about 58 mol %, from about 52 mol % toabout 57 mol %, from about 52 mol % to about 56 mol %, or from about 53mol % to about 55 mol % (or any fraction thereof or range therein) ofthe total lipid present in the particle. In particular embodiments, thecationic lipid comprises about 50 mol %, 51 mol %, 52 mol %, 53 mol %,54 mol %, 55 mol %, 56 mol %, 57 mol %, 58 mol %, 59 mol %, 60 mol %, 61mol %, 62 mol %, 63 mol %, 64 mol %, or 65 mol % (or any fractionthereof or range therein) of the total lipid present in the particle. Inother embodiments, the cationic lipid comprises (at least) about 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, or 90 mol % (or any fraction thereof or range therein)of the total lipid present in the particle.

In additional embodiments, the cationic lipid comprises from about 2 mol% to about 60 mol %, from about 5 mol % to about 50 mol %, from about 10mol % to about 50 mol %, from about 20 mol % to about 50 mol %, fromabout 20 mol % to about 40 mol %, from about 30 mol % to about 40 mol %,or about 40 mol % (or any fraction thereof or range therein) of thetotal lipid present in the particle.

Additional percentages and ranges of cationic lipids suitable for use inthe lipid particles of the present invention are described in PCTPublication No. WO 09/127060, U.S. Publication No. 20110071208, and U.S.Publication No. 20110076335, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

It should be understood that the percentage of cationic lipid present inthe lipid particles of the invention is a target amount, and that theactual amount of cationic lipid present in the formulation may vary, forexample, by +5 mol %. For example, in the 1:57 lipid particle (e.g.,SNALP) formulation, the target amount of cationic lipid is 57.1 mol %,but the actual amount of cationic lipid may be ±5 mol %, ±4 mol %, ±3mol %, ±2 mol %, ±1 mol %, ±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or +0.1mol % of that target amount, with the balance of the formulation beingmade up of other lipid components (adding up to 100 mol % of totallipids present in the particle). Similarly, in the 7:54 lipid particle(e.g., SNALP) formulation, the target amount of cationic lipid is 54.06mol %, but the actual amount of cationic lipid may be ±5 mol %, +4 mol%, ±3 mol %, ±2 mol %, ±1 mol %, +0.75 mol %, ±0.5 mol %, ±0.25 mol %,or ±0.1 mol % of that target amount, with the balance of the formulationbeing made up of other lipid components (adding up to 100 mol % of totallipids present in the particle).

B. Non-Cationic Lipids

The non-cationic lipids used in the lipid particles of the invention(e.g., SNALP) can be any of a variety of neutral uncharged,zwitterionic, or anionic lipids capable of producing a stable complex.

Non-limiting examples of non-cationic lipids include phospholipids suchas lecithin, phosphatidylethanolamine, lysolecithin,lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin,phosphatidic acid, cerebrosides, dicetylphosphate,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoyl-phosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),palmitoyloleyol-phosphatidylglycerol (POPG),dioleoylphosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),dipalmitoyl-phosphatidylethanolamine (DPPE),dimyristoyl-phosphatidylethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE),monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine,dielaidoyl-phosphatidylethanolamine (DEPE),stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine,dilinoleoylphosphatidylcholine, and mixtures thereof. Otherdiacylphosphatidylcholine and diacylphosphatidylethanolaminephospholipids can also be used. The acyl groups in these lipids arepreferably acyl groups derived from fatty acids having C₁₀-C₂₄ carbonchains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

Additional examples of non-cationic lipids include sterols such ascholesterol and derivatives thereof. Non-limiting examples ofcholesterol derivatives include polar analogues such as 5α-cholestanol,5β-coprostanol, cholesteryl-(2′-hydroxy)-ethyl ether,cholesteryl-(4′-hydroxy)-butyl ether, and 6-ketocholestanol; non-polaranalogues such as 5α-cholestane, cholestenone, 5α-cholestanone,5β-cholestanone, and cholesteryl decanoate; and mixtures thereof. Inpreferred embodiments, the cholesterol derivative is a polar analoguesuch as cholesteryl-(4′-hydroxy)-butyl ether. The synthesis ofcholesteryl-(2′-hydroxy)-ethyl ether is described in PCT Publication No.WO 09/127060, the disclosure of which is herein incorporated byreference in its entirety for all purposes.

In some embodiments, the non-cationic lipid present in the lipidparticles (e.g., SNALP) comprises or consists of a mixture of one ormore phospholipids and cholesterol or a derivative thereof. In otherembodiments, the non-cationic lipid present in the lipid particles(e.g., SNALP) comprises or consists of one or more phospholipids, e.g.,a cholesterol-free lipid particle formulation. In yet other embodiments,the non-cationic lipid present in the lipid particles (e.g., SNALP)comprises or consists of cholesterol or a derivative thereof, e.g., aphospholipid-free lipid particle formulation.

Other examples of non-cationic lipids suitable for use in the presentinvention include nonphosphorous containing lipids such as, e.g.,stearylamine, dodecylamine, hexadecylamine, acetyl palmitate,glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphotericacrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfatepolyethyloxylated fatty acid amides, dioctadecyldimethyl ammoniumbromide, ceramide, sphingomyelin, and the like.

In some embodiments, the non-cationic lipid comprises from about 10 mol% to about 60 mol %, from about 20 mol % to about 55 mol %, from about20 mol % to about 45 mol %, from about 20 mol % to about 40 mol %, fromabout 25 mol % to about 50 mol %, from about 25 mol % to about 45 mol %,from about 30 mol % to about 50 mol %, from about 30 mol % to about 45mol %, from about 30 mol % to about 40 mol %, from about 35 mol % toabout 45 mol %, from about 37 mol % to about 42 mol %, or about 35 mol%, 36 mol %, 37 mol %, 38 mol %, 39 mol %, 40 mol %, 41 mol %, 42 mol %,43 mol %, 44 mol %, or 45 mol % (or any fraction thereof or rangetherein) of the total lipid present in the particle.

In embodiments where the lipid particles contain a mixture ofphospholipid and cholesterol or a cholesterol derivative, the mixturemay comprise up to about 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60mol % of the total lipid present in the particle.

In some embodiments, the phospholipid component in the mixture maycomprise from about 2 mol % to about 20 mol %, from about 2 mol % toabout 15 mol %, from about 2 mol % to about 12 mol %, from about 4 mol %to about 15 mol %, or from about 4 mol % to about 10 mol % (or anyfraction thereof or range therein) of the total lipid present in theparticle. In certain preferred embodiments, the phospholipid componentin the mixture comprises from about 5 mol % to about 10 mol %, fromabout 5 mol % to about 9 mol %, from about 5 mol % to about 8 mol %,from about 6 mol % to about 9 mol %, from about 6 mol % to about 8 mol%, or about 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (orany fraction thereof or range therein) of the total lipid present in theparticle. As a non-limiting example, a 1:57 lipid particle formulationcomprising a mixture of phospholipid and cholesterol may comprise aphospholipid such as DPPC or DSPC at about 7 mol % (or any fractionthereof), e.g., in a mixture with cholesterol or a cholesterolderivative at about 34 mol % (or any fraction thereof) of the totallipid present in the particle. As another non-limiting example, a 7:54lipid particle formulation comprising a mixture of phospholipid andcholesterol may comprise a phospholipid such as DPPC or DSPC at about 7mol % (or any fraction thereof), e.g., in a mixture with cholesterol ora cholesterol derivative at about 32 mol % (or any fraction thereof) ofthe total lipid present in the particle.

In other embodiments, the cholesterol component in the mixture maycomprise from about 25 mol % to about 45 mol %, from about 25 mol % toabout 40 mol %, from about 30 mol % to about 45 mol %, from about 30 mol% to about 40 mol %, from about 27 mol % to about 37 mol %, from about25 mol % to about 30 mol %, or from about 35 mol % to about 40 mol % (orany fraction thereof or range therein) of the total lipid present in theparticle. In certain preferred embodiments, the cholesterol component inthe mixture comprises from about 25 mol % to about 35 mol %, from about27 mol % to about 35 mol %, from about 29 mol % to about 35 mol %, fromabout 30 mol % to about 35 mol %, from about 30 mol % to about 34 mol %,from about 31 mol % to about 33 mol %, or about 30 mol %, 31 mol %, 32mol %, 33 mol %, 34 mol %, or 35 mol % (or any fraction thereof or rangetherein) of the total lipid present in the particle. In otherembodiments, the cholesterol component in the mixture comprises about36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 mol % (or any fraction thereofor range therein) of the total lipid present in the particle. Typically,a 1:57 lipid particle formulation comprising a mixture of phospholipidand cholesterol may comprise cholesterol or a cholesterol derivative atabout 34 mol % (or any fraction thereof), e.g., in a mixture with aphospholipid such as DPPC or DSPC at about 7 mol % (or any fractionthereof) of the total lipid present in the particle. Typically, a 7:54lipid particle formulation comprising a mixture of phospholipid andcholesterol may comprise cholesterol or a cholesterol derivative atabout 32 mol % (or any fraction thereof), e.g., in a mixture with aphospholipid such as DPPC or DSPC at about 7 mol % (or any fractionthereof) of the total lipid present in the particle.

In embodiments where the lipid particles are phospholipid-free, thecholesterol or derivative thereof may comprise up to about 25 mol %, 30mol %, 35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60 mol % ofthe total lipid present in the particle.

In some embodiments, the cholesterol or derivative thereof in thephospholipid-free lipid particle formulation may comprise from about 25mol % to about 45 mol %, from about 25 mol % to about 40 mol %, fromabout 30 mol % to about 45 mol %, from about 30 mol % to about 40 mol %,from about 31 mol % to about 39 mol %, from about 32 mol % to about 38mol %, from about 33 mol % to about 37 mol %, from about 35 mol % toabout 45 mol %, from about 30 mol % to about 35 mol %, from about 35 mol% to about 40 mol %, or about 30 mol %, 31 mol %, 32 mol %, 33 mol %, 34mol %, 35 mol %, 36 mol %, 37 mol %, 38 mol %, 39 mol %, 40 mol %, 41mol %, 42 mol %, 43 mol %, 44 mol %, or 45 mol % (or any fractionthereof or range therein) of the total lipid present in the particle. Asa non-limiting example, a 1:62 lipid particle formulation may comprisecholesterol at about 37 mol % (or any fraction thereof) of the totallipid present in the particle. As another non-limiting example, a 7:58lipid particle formulation may comprise cholesterol at about 35 mol %(or any fraction thereof) of the total lipid present in the particle.

In other embodiments, the non-cationic lipid comprises from about 5 mol% to about 90 mol %, from about 10 mol % to about 85 mol %, from about20 mol % to about 80 mol %, about 10 mol % (e.g., phospholipid only), orabout 60 mol % (e.g., phospholipid and cholesterol or derivativethereof) (or any fraction thereof or range therein) of the total lipidpresent in the particle.

Additional percentages and ranges of non-cationic lipids suitable foruse in the lipid particles of the present invention are described in PCTPublication No. WO 09/127060, U.S. Publication No. 20110071208, and U.S.Publication No. 20110076335, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

It should be understood that the percentage of non-cationic lipidpresent in the lipid particles of the invention is a target amount, andthat the actual amount of non-cationic lipid present in the formulationmay vary, for example, by ±5 mol %. For example, in the 1:57 lipidparticle (e.g., SNALP) formulation, the target amount of phospholipid is7.1 mol % and the target amount of cholesterol is 34.3 mol %, but theactual amount of phospholipid may be ±2 mol %, ±1.5 mol %, ±1 mol %,±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol % of that targetamount, and the actual amount of cholesterol may be ±3 mol %, ±2 mol %,±1 mol %, ±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol % of thattarget amount, with the balance of the formulation being made up ofother lipid components (adding up to 100 mol % of total lipids presentin the particle). Similarly, in the 7:54 lipid particle (e.g., SNALP)formulation, the target amount of phospholipid is 6.75 mol % and thetarget amount of cholesterol is 32.43 mol %, but the actual amount ofphospholipid may be ±2 mol %, ±1.5 mol %, ±1 mol %, ±0.75 mol %, ±0.5mol %, ±0.25 mol %, or ±0.1 mol % of that target amount, and the actualamount of cholesterol may be ±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol %,±0.5 mol %, ±0.25 mol %, or ±0.1 mol % of that target amount, with thebalance of the formulation being made up of other lipid components(adding up to 100 mol % of total lipids present in the particle).

C. Lipid Conjugates

In addition to cationic and non-cationic lipids, the lipid particles ofthe invention (e.g., SNALP) may further comprise a lipid conjugate. Theconjugated lipid is useful in that it prevents the aggregation ofparticles. Suitable conjugated lipids include, but are not limited to,PEG-lipid conjugates, POZ-lipid conjugates, ATTA-lipid conjugates,cationic-polymer-lipid conjugates (CPLs), and mixtures thereof. Incertain embodiments, the lipid particles comprise either a PEG-lipidconjugate or an ATTA-lipid conjugate together with a CPL. The term“ATTA” or “polyamide” includes, without limitation, compounds describedin U.S. Pat. Nos. 6,320,017 and 6,586,559, the disclosures of which areherein incorporated by reference in their entirety for all purposes.

In a preferred embodiment, the lipid conjugate is a PEG-lipid. Examplesof PEG-lipids include, but are not limited to, PEG coupled todialkyloxypropyls (PEG-DAA) as described in, e.g., PCT Publication No.WO 05/026372, PEG coupled to diacylglycerol (PEG-DAG) as described in,e.g., U.S. Patent Publication Nos. 20030077829 and 2005008689, PEGcoupled to phospholipids such as phosphatidylethanolamine (PEG-PE), PEGconjugated to ceramides as described in, e.g., U.S. Pat. No. 5,885,613,PEG conjugated to cholesterol or a derivative thereof, and mixturesthereof. The disclosures of these patent documents are hereinincorporated by reference in their entirety for all purposes.

Additional PEG-lipids suitable for use in the invention include, withoutlimitation, mPEG2000-1,2-di-O-alkyl-sn3-carbomoylglyceride (PEG-C-DOMG).The synthesis of PEG-C-DOMG is described in PCT Publication No. WO09/086558, the disclosure of which is herein incorporated by referencein its entirety for all purposes. Yet additional suitable PEG-lipidconjugates include, without limitation,1-[8′-(1,2-dimyristoyl-3-propanoxy)-carboxamido-3′,6′-dioxaoctanyl]carbamoyl-o-methyl-poly(ethyleneglycol) (2KPEG-DMG). The synthesis of 2KPEG-DMG is described in U.S.Pat. No. 7,404,969, the disclosure of which is herein incorporated byreference in its entirety for all purposes.

PEG is a linear, water-soluble polymer of ethylene PEG repeating unitswith two terminal hydroxyl groups. PEGs are classified by theirmolecular weights; for example, PEG 2000 has an average molecular weightof about 2,000 daltons, and PEG 5000 has an average molecular weight ofabout 5,000 daltons. PEGs are commercially available from Sigma ChemicalCo. and other companies and include, but are not limited to, thefollowing: monomethoxypolyethylene glycol (MePEG-OH),monomethoxypolyethylene glycol-succinate (MePEG-S),monomethoxypolyethylene glycol-succinimidyl succinate (MePEG-S-NHS),monomethoxypolyethylene glycol-amine (MePEG-NH₂),monomethoxypolyethylene glycol-tresylate (MePEG-TRES),monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM), as wellas such compounds containing a terminal hydroxyl group instead of aterminal methoxy group (e.g., HO-PEG-S, HO-PEG-S-NHS, HO-PEG-NH₂, etc.).Other PEGs such as those described in U.S. Pat. Nos. 6,774,180 and7,053,150 (e.g., mPEG (20 KDa) amine) are also useful for preparing thePEG-lipid conjugates of the present invention. The disclosures of thesepatents are herein incorporated by reference in their entirety for allpurposes. In addition, monomethoxypolyethyleneglycol-acetic acid(MePEG-CH₂COOH) is particularly useful for preparing PEG-lipidconjugates including, e.g., PEG-DAA conjugates.

The PEG moiety of the PEG-lipid conjugates described herein may comprisean average molecular weight ranging from about 550 daltons to about10,000 daltons. In certain instances, the PEG moiety has an averagemolecular weight of from about 750 daltons to about 5,000 daltons (e.g.,from about 1,000 daltons to about 5,000 daltons, from about 1,500daltons to about 3,000 daltons, from about 750 daltons to about 3,000daltons, from about 750 daltons to about 2,000 daltons, etc.). In otherinstances, the PEG moiety has an average molecular weight of from about550 daltons to about 1000 daltons, from about 250 daltons to about 1000daltons, from about 400 daltons to about 1000 daltons, from about 600daltons to about 900 daltons, from about 700 daltons to about 800daltons, or about 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, or 1000 daltons. In preferred embodiments, thePEG moiety has an average molecular weight of about 2,000 daltons orabout 750 daltons.

In certain instances, the PEG can be optionally substituted by an alkyl,alkoxy, acyl, or aryl group. The PEG can be conjugated directly to thelipid or may be linked to the lipid via a linker moiety. Any linkermoiety suitable for coupling the PEG to a lipid can be used including,e.g., non-ester containing linker moieties and ester-containing linkermoieties. In a preferred embodiment, the linker moiety is a non-estercontaining linker moiety. As used herein, the term “non-ester containinglinker moiety” refers to a linker moiety that does not contain acarboxylic ester bond (—OC(O)—). Suitable non-ester containing linkermoieties include, but are not limited to, amido (—C(O)NH—), amino(—NR—), carbonyl (—C(O)—), carbamate (—NHC(O)O—), urea (—NHC(O)NH—),disulphide (—S—S—), ether (—O—), succinyl (—(O)CCH₂CH₂C(O)—),succinamidyl (—NHC(O)CH₂CH₂C(O)NH—), ether, disulphide, as well ascombinations thereof (such as a linker containing both a carbamatelinker moiety and an amido linker moiety). In a preferred embodiment, acarbamate linker is used to couple the PEG to the lipid.

In other embodiments, an ester containing linker moiety is used tocouple the PEG to the lipid. Suitable ester containing linker moietiesinclude, e.g., carbonate (—OC(O)O—), succinoyl, phosphate esters(—O—(O)POH—O—), sulfonate esters, and combinations thereof.

Phosphatidylethanolamines having a variety of acyl chain groups ofvarying chain lengths and degrees of saturation can be conjugated to PEGto form the lipid conjugate. Such phosphatidylethanolamines arecommercially available, or can be isolated or synthesized usingconventional techniques known to those of skilled in the art.Phosphatidylethanolamines containing saturated or unsaturated fattyacids with carbon chain lengths in the range of C₁₀ to C₂₀ arepreferred. Phosphatidylethanolamines with mono- or diunsaturated fattyacids and mixtures of saturated and unsaturated fatty acids can also beused. Suitable phosphatidylethanolamines include, but are not limitedto, dimyristoyl-phosphatidylethanolamine (DMPE),dipalmitoyl-phosphatidylethanolamine (DPPE),dioleoylphosphatidylethanolamine (DOPE), anddistearoyl-phosphatidylethanolamine (DSPE).

The term “diacylglycerol” or “DAG” includes a compound having 2 fattyacyl chains, R¹ and R², both of which have independently between 2 and30 carbons bonded to the 1- and 2-position of glycerol by esterlinkages. The acyl groups can be saturated or have varying degrees ofunsaturation. Suitable acyl groups include, but are not limited to,lauroyl (C₁₂), myristoyl (C₁₄), palmitoyl (C₁₆), stearoyl (C₁₈), andicosoyl (C₂₀). In preferred embodiments, R¹ and R² are the same, i.e.,R¹ and R² are both myristoyl (i.e., dimyristoyl), R¹ and R² are bothstearoyl (i.e., distearoyl), etc. Diacylglycerols have the followinggeneral formula:

The term “dialkyloxypropyl” or “DAA” includes a compound having 2 alkylchains, R¹ and R², both of which have independently between 2 and 30carbons. The alkyl groups can be saturated or have varying degrees ofunsaturation. Dialkyloxypropyls have the following general formula:

In a preferred embodiment, the PEG-lipid is a PEG-DAA conjugate havingthe following formula:

wherein R¹ and R² are independently selected and are long-chain alkylgroups having from about 10 to about 22 carbon atoms; PEG is apolyethyleneglycol; and L is a non-ester containing linker moiety or anester containing linker moiety as described above. The long-chain alkylgroups can be saturated or unsaturated. Suitable alkyl groups include,but are not limited to, decyl (C₁₀), lauryl (C₁₂), myristyl (C₁₄),palmityl (C₁₆), stearyl (C₁₈), and icosyl (C₂₀). In preferredembodiments, R¹ and R² are the same, i.e., R¹ and R² are both myristyl(i.e., dimyristyl), R¹ and R² are both stearyl (i.e., distearyl), etc.

In Formula VI above, the PEG has an average molecular weight rangingfrom about 550 daltons to about 10,000 daltons. In certain instances,the PEG has an average molecular weight of from about 750 daltons toabout 5,000 daltons (e.g., from about 1,000 daltons to about 5,000daltons, from about 1,500 daltons to about 3,000 daltons, from about 750daltons to about 3,000 daltons, from about 750 daltons to about 2,000daltons, etc.). In other instances, the PEG moiety has an averagemolecular weight of from about 550 daltons to about 1000 daltons, fromabout 250 daltons to about 1000 daltons, from about 400 daltons to about1000 daltons, from about 600 daltons to about 900 daltons, from about700 daltons to about 800 daltons, or about 200, 250, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 daltons. Inpreferred embodiments, the PEG has an average molecular weight of about2,000 daltons or about 750 daltons. The PEG can be optionallysubstituted with alkyl, alkoxy, acyl, or aryl groups. In certainembodiments, the terminal hydroxyl group is substituted with a methoxyor methyl group.

In a preferred embodiment, “L” is a non-ester containing linker moiety.Suitable non-ester containing linkers include, but are not limited to,an amido linker moiety, an amino linker moiety, a carbonyl linkermoiety, a carbamate linker moiety, a urea linker moiety, an ether linkermoiety, a disulphide linker moiety, a succinamidyl linker moiety, andcombinations thereof. In a preferred embodiment, the non-estercontaining linker moiety is a carbamate linker moiety (i.e., a PEG-C-DAAconjugate). In another preferred embodiment, the non-ester containinglinker moiety is an amido linker moiety (i.e., a PEG-A-DAA conjugate).In yet another preferred embodiment, the non-ester containing linkermoiety is a succinamidyl linker moiety (i.e., a PEG-S-DAA conjugate).

In particular embodiments, the PEG-lipid conjugate is selected from:

The PEG-DAA conjugates are synthesized using standard techniques andreagents known to those of skill in the art. It will be recognized thatthe PEG-DAA conjugates will contain various amide, amine, ether, thio,carbamate, and urea linkages. Those of skill in the art will recognizethat methods and reagents for forming these bonds are well known andreadily available. See, e.g., March, ADVANCED ORGANIC CHEMISTRY (Wiley1992); Larock, COMPREHENSIVE ORGANIC TRANSFORMATIONS (VCH 1989); andFurniss, VOGEL'S TEXTBOOK OF PRACTICAL ORGANIC CHEMISTRY, 5th ed.(Longman 1989). It will also be appreciated that any functional groupspresent may require protection and deprotection at different points inthe synthesis of the PEG-DAA conjugates. Those of skill in the art willrecognize that such techniques are well known. See, e.g., Green andWuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS (Wiley 1991).

Preferably, the PEG-DAA conjugate is a PEG-didecyloxypropyl (C₁₀)conjugate, a PEG-dilauryloxypropyl (C₁₂) conjugate, aPEG-dimyristyloxypropyl (C₁₄) conjugate, a PEG-dipalmityloxypropyl (C₁₆)conjugate, or a PEG-distearyloxypropyl (C₁₈) conjugate. In theseembodiments, the PEG preferably has an average molecular weight of about750 or about 2,000 daltons. In one particularly preferred embodiment,the PEG-lipid conjugate comprises PEG2000-C-DMA, wherein the “2000”denotes the average molecular weight of the PEG, the “C” denotes acarbamate linker moiety, and the “DMA” denotes dimyristyloxypropyl. Inanother particularly preferred embodiment, the PEG-lipid conjugatecomprises PEG750-C-DMA, wherein the “750” denotes the average molecularweight of the PEG, the “C” denotes a carbamate linker moiety, and the“DMA” denotes dimyristyloxypropyl. In particular embodiments, theterminal hydroxyl group of the PEG is substituted with a methyl group.Those of skill in the art will readily appreciate that otherdialkyloxypropyls can be used in the PEG-DAA conjugates of the presentinvention.

In addition to the foregoing, it will be readily apparent to those ofskill in the art that other hydrophilic polymers can be used in place ofPEG. Examples of suitable polymers that can be used in place of PEGinclude, but are not limited to, polyvinylpyrrolidone,polymethyloxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide and polydimethylacrylamide,polylactic acid, polyglycolic acid, and derivatized celluloses such ashydroxymethylcellulose or hydroxyethylcellulose.

In addition to the foregoing components, the lipid particles (e.g.,SNALP) of the present invention can further comprise cationicpoly(ethylene glycol) (PEG) lipids or CPLs (see, e.g., Chen et al.,Bioconj. Chem., 11:433-437 (2000); U.S. Pat. No. 6,852,334; PCTPublication No. WO 00/62813, the disclosures of which are hereinincorporated by reference in their entirety for all purposes).

In some embodiments, the lipid conjugate (e.g., PEG-lipid) comprisesfrom about 0.1 mol % to about 2 mol %, from about 0.5 mol % to about 2mol %, from about 1 mol % to about 2 mol %, from about 0.6 mol % toabout 1.9 mol %, from about 0.7 mol % to about 1.8 mol %, from about 0.8mol % to about 1.7 mol %, from about 0.9 mol % to about 1.6 mol %, fromabout 0.9 mol % to about 1.8 mol %, from about 1 mol % to about 1.8 mol%, from about 1 mol % to about 1.7 mol %, from about 1.2 mol % to about1.8 mol %, from about 1.2 mol % to about 1.7 mol %, from about 1.3 mol %to about 1.6 mol %, from about 1.4 mol % to about 1.5 mol %, or about 1,1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mol % (or any fractionthereof or range therein) of the total lipid present in the particle.

In other embodiments, the lipid conjugate (e.g., PEG-lipid) comprisesfrom about 0 mol % to about 20 mol %, from about 0.5 mol % to about 20mol %, from about 2 mol % to about 20 mol %, from about 1.5 mol % toabout 18 mol %, from about 2 mol % to about 15 mol %, from about 4 mol %to about 15 mol %, from about 2 mol % to about 12 mol %, from about 5mol % to about 12 mol %, or about 2 mol % (or any fraction thereof orrange therein) of the total lipid present in the particle.

In further embodiments, the lipid conjugate (e.g., PEG-lipid) comprisesfrom about 4 mol % to about 10 mol %, from about 5 mol % to about 10 mol%, from about 5 mol % to about 9 mol %, from about 5 mol % to about 8mol %, from about 6 mol % to about 9 mol %, from about 6 mol % to about8 mol %, or about 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol% (or any fraction thereof or range therein) of the total lipid presentin the particle.

Additional examples, percentages, and/or ranges of lipid conjugatessuitable for use in the lipid particles of the invention are describedin PCT Publication No. WO 09/127060, U.S. Publication No. 20110071208,U.S. Publication No. 20110076335, U.S. application Ser. No. 13/006,277,filed Jan. 13, 2011, and PCT Publication No. WO 2010/006282, thedisclosures of which are herein incorporated by reference in theirentirety for all purposes.

It should be understood that the percentage of lipid conjugate (e.g.,PEG-lipid) present in the lipid particles of the invention is a targetamount, and that the actual amount of lipid conjugate present in theformulation may vary, for example, by ±2 mol %. For example, in the 1:57lipid particle (e.g., SNALP) formulation, the target amount of lipidconjugate is 1.4 mol %, but the actual amount of lipid conjugate may be±0.5 mol %, ±0.4 mol %, ±0.3 mol %, ±0.2 mol %, ±0.1 mol %, or ±0.05 mol% of that target amount, with the balance of the formulation being madeup of other lipid components (adding up to 100 mol % of total lipidspresent in the particle). Similarly, in the 7:54 lipid particle (e.g.,SNALP) formulation, the target amount of lipid conjugate is 6.76 mol %,but the actual amount of lipid conjugate may be ±2 mol %, ±1.5 mol %, ±1mol %, ±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol % of thattarget amount, with the balance of the formulation being made up ofother lipid components (adding up to 100 mol % of total lipids presentin the particle).

One of ordinary skill in the art will appreciate that the concentrationof the lipid conjugate can be varied depending on the lipid conjugateemployed and the rate at which the lipid particle is to becomefusogenic.

By controlling the composition and concentration of the lipid conjugate,one can control the rate at which the lipid conjugate exchanges out ofthe lipid particle and, in turn, the rate at which the lipid particlebecomes fusogenic. For instance, when a PEG-DAA conjugate is used as thelipid conjugate, the rate at which the lipid particle becomes fusogeniccan be varied, for example, by varying the concentration of the lipidconjugate, by varying the molecular weight of the PEG, or by varying thechain length and degree of saturation of the alkyl groups on the PEG-DAAconjugate. In addition, other variables including, for example, pH,temperature, ionic strength, etc. can be used to vary and/or control therate at which the lipid particle becomes fusogenic. Other methods whichcan be used to control the rate at which the lipid particle becomesfusogenic will become apparent to those of skill in the art upon readingthis disclosure. Also, by controlling the composition and concentrationof the lipid conjugate, one can control the lipid particle (e.g., SNALP)size.

VI. Preparation of Lipid Particles

The lipid particles of the present invention, e.g., SNALP, in which anactive agent such as a nucleic acid (e.g., an interfering RNA such as ansiRNA) is entrapped within the lipid portion of the particle and isprotected from degradation, can be formed by any method known in the artincluding, but not limited to, a continuous mixing method, a directdilution process, and an in-line dilution process. In certainembodiments, one or more antioxidants such as metal chelators (e.g.,EDTA), primary antioxidants, and/or secondary antioxidants may beincluded at any step or at multiple steps in the process (e.g., priorto, during, and/or after lipid particle formation) as described in PCTApplication No. PCT/CA2010/001919, the disclosure of which is hereinincorporated by reference in its entirety for all purposes.

In particular embodiments, the cationic lipids may comprise at leastone, two, three, four, five, or more cationic lipids such as those setforth in Formulas I-III or salts thereof, alone or in combination withother cationic lipid species. In other embodiments, the non-cationiclipids may comprise one, two, or more lipids including egg sphingomyelin(ESM), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC),dipalmitoyl-phosphatidylcholine (DPPC),monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine,14:0 PE (1,2-dimyristoyl-phosphatidylethanolamine (DMPE)), 16:0 PE(1,2-dipalmitoyl-phosphatidylethanolamine (DPPE)), 18:0 PE(1,2-distearoyl-phosphatidylethanolamine (DSPE)), 18:1 PE(1,2-dioleoylphosphatidylethanolamine (DOPE)), 18:1 trans PE(1,2-dielaidoyl-phosphatidylethanolamine (DEPE)), 18:0-18:1 PE(1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE)), 16:0-18:1 PE(1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE)), polyethyleneglycol-based polymers (e.g., PEG 2000, PEG 5000, PEG-modifieddiacylglycerols, or PEG-modified dialkyloxypropyls), cholesterol,derivatives thereof, or combinations thereof.

In certain embodiments, the present invention provides nucleicacid-lipid particles (e.g., SNALP) produced via a continuous mixingmethod, e.g., a process that includes providing an aqueous solutioncomprising a nucleic acid (e.g., interfering RNA) in a first reservoir,providing an organic lipid solution in a second reservoir (wherein thelipids present in the organic lipid solution are solubilized in anorganic solvent, e.g., a lower alkanol such as ethanol), and mixing theaqueous solution with the organic lipid solution such that the organiclipid solution mixes with the aqueous solution so as to substantiallyinstantaneously produce a lipid vesicle (e.g., liposome) encapsulatingthe nucleic acid within the lipid vesicle. This process and theapparatus for carrying out this process are described in detail in U.S.Patent Publication No. 20040142025, the disclosure of which is hereinincorporated by reference in its entirety for all purposes.

The action of continuously introducing lipid and buffer solutions into amixing environment, such as in a mixing chamber, causes a continuousdilution of the lipid solution with the buffer solution, therebyproducing a lipid vesicle substantially instantaneously upon mixing. Asused herein, the phrase “continuously diluting a lipid solution with abuffer solution” (and variations) generally means that the lipidsolution is diluted sufficiently rapidly in a hydration process withsufficient force to effectuate vesicle generation. By mixing the aqueoussolution comprising a nucleic acid with the organic lipid solution, theorganic lipid solution undergoes a continuous stepwise dilution in thepresence of the buffer solution (i.e., aqueous solution) to produce anucleic acid-lipid particle.

The nucleic acid-lipid particles formed using the continuous mixingmethod typically have a size of from about 30 nm to about 150 nm, fromabout 40 nm to about 150 nm, from about 50 nm to about 150 nm, fromabout 60 nm to about 130 nm, from about 70 nm to about 110 nm, fromabout 70 nm to about 100 nm, from about 80 nm to about 100 nm, fromabout 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80nm to about 90 nm, from about 70 nm to about 80 nm, less than about 120nm, 110 nm, 100 nm, 90 nm, or 80 nm, or about 30 nm, 35 nm, 40 nm, 45nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140nm, 145 nm, or 150 nm (or any fraction thereof or range therein). Theparticles thus formed do not aggregate and are optionally sized toachieve a uniform particle size.

In another embodiment, the present invention provides nucleic acid-lipidparticles (e.g., SNALP) produced via a direct dilution process thatincludes forming a lipid vesicle (e.g., liposome) solution andimmediately and directly introducing the lipid vesicle solution into acollection vessel containing a controlled amount of dilution buffer. Inpreferred aspects, the collection vessel includes one or more elementsconfigured to stir the contents of the collection vessel to facilitatedilution. In one aspect, the amount of dilution buffer present in thecollection vessel is substantially equal to the volume of lipid vesiclesolution introduced thereto. As a non-limiting example, a lipid vesiclesolution in 45% ethanol when introduced into the collection vesselcontaining an equal volume of dilution buffer will advantageously yieldsmaller particles.

In yet another embodiment, the present invention provides nucleicacid-lipid particles (e.g., SNALP) produced via an in-line dilutionprocess in which a third reservoir containing dilution buffer is fluidlycoupled to a second mixing region. In this embodiment, the lipid vesicle(e.g., liposome) solution formed in a first mixing region is immediatelyand directly mixed with dilution buffer in the second mixing region. Inpreferred aspects, the second mixing region includes a T-connectorarranged so that the lipid vesicle solution and the dilution bufferflows meet as opposing 180° flows; however, connectors providingshallower angles can be used, e.g., from about 27° to about 180° (e.g.,about 90°). A pump mechanism delivers a controllable flow of buffer tothe second mixing region. In one aspect, the flow rate of dilutionbuffer provided to the second mixing region is controlled to besubstantially equal to the flow rate of lipid vesicle solutionintroduced thereto from the first mixing region. This embodimentadvantageously allows for more control of the flow of dilution buffermixing with the lipid vesicle solution in the second mixing region, andtherefore also the concentration of lipid vesicle solution in bufferthroughout the second mixing process. Such control of the dilutionbuffer flow rate advantageously allows for small particle size formationat reduced concentrations.

These processes and the apparatuses for carrying out these directdilution and in-line dilution processes are described in detail in U.S.Patent Publication No. 20070042031, the disclosure of which is hereinincorporated by reference in its entirety for all purposes.

The nucleic acid-lipid particles formed using the direct dilution andin-line dilution processes typically have a size of from about 30 nm toabout 150 nm, from about 40 nm to about 150 nm, from about 50 nm toabout 150 nm, from about 60 nm to about 130 nm, from about 70 nm toabout 110 nm, from about 70 nm to about 100 nm, from about 80 nm toabout 100 nm, from about 90 nm to about 100 nm, from about 70 to about90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm,less than about 120 nm, 110 nm, 100 nm, 90 nm, or 80 nm, or about 30 nm,35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130nm, 135 nm, 140 nm, 145 nm, or 150 nm. or any fraction thereof or rangetherein). The particles thus formed do not aggregate and are optionallysized to achieve a uniform particle size.

If needed, the lipid particles of the invention (e.g., SNALP) can besized by any of the methods available for sizing liposomes. The sizingmay be conducted in order to achieve a desired size range and relativelynarrow distribution of particle sizes.

Several techniques are available for sizing the particles to a desiredsize. One sizing method, used for liposomes and equally applicable tothe present particles, is described in U.S. Pat. No. 4,737,323, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes. Sonicating a particle suspension either by bath orprobe sonication produces a progressive size reduction down to particlesof less than about 50 nm in size. Homogenization is another method whichrelies on shearing energy to fragment larger particles into smallerones. In a typical homogenization procedure, particles are recirculatedthrough a standard emulsion homogenizer until selected particle sizes,typically between about 60 and about 80 nm, are observed. In bothmethods, the particle size distribution can be monitored by conventionallaser-beam particle size discrimination, or QELS.

Extrusion of the particles through a small-pore polycarbonate membraneor an asymmetric ceramic membrane is also an effective method forreducing particle sizes to a relatively well-defined size distribution.Typically, the suspension is cycled through the membrane one or moretimes until the desired particle size distribution is achieved. Theparticles may be extruded through successively smaller-pore membranes,to achieve a gradual reduction in size.

In some embodiments, the nucleic acids present in the particles areprecondensed as described in, e.g., U.S. patent application Ser. No.09/744,103, the disclosure of which is herein incorporated by referencein its entirety for all purposes.

In other embodiments, the methods may further comprise adding non-lipidpolycations which are useful to effect the lipofection of cells usingthe present compositions. Examples of suitable non-lipid polycationsinclude, hexadimethrine bromide (sold under the brand name POLYBRENE®,from Aldrich Chemical Co., Milwaukee, Wis., USA) or other salts ofhexadimethrine. Other suitable polycations include, for example, saltsof poly-L-ornithine, poly-L-arginine, poly-L-lysine, poly-D-lysine,polyallylamine, and polyethyleneimine. Addition of these salts ispreferably after the particles have been formed.

In some embodiments, the nucleic acid to lipid ratios (mass/mass ratios)in a formed nucleic acid-lipid particle (e.g., SNALP) will range fromabout 0.01 to about 0.2, from about 0.05 to about 0.2, from about 0.02to about 0.1, from about 0.03 to about 0.1, or from about 0.01 to about0.08. The ratio of the starting materials (input) also falls within thisrange. In other embodiments, the particle preparation uses about 400 μgnucleic acid per 10 mg total lipid or a nucleic acid to lipid mass ratioof about 0.01 to about 0.08 and, more preferably, about 0.04, whichcorresponds to 1.25 mg of total lipid per 50 μg of nucleic acid. Inother preferred embodiments, the particle has a nucleic acid:lipid massratio of about 0.08.

In other embodiments, the lipid to nucleic acid ratios (mass/massratios) in a formed nucleic acid-lipid particle (e.g., SNALP) will rangefrom about 1 (1:1) to about 100 (100:1), from about 5 (5:1) to about 100(100:1), from about 1 (1:1) to about 50 (50:1), from about 2 (2:1) toabout 50 (50:1), from about 3 (3:1) to about 50 (50:1), from about 4(4:1) to about 50 (50:1), from about 5 (5:1) to about 50 (50:1), fromabout 1 (1:1) to about 25 (25:1), from about 2 (2:1) to about 25 (25:1),from about 3 (3:1) to about 25 (25:1), from about 4 (4:1) to about 25(25:1), from about 5 (5:1) to about 25 (25:1), from about 5 (5:1) toabout 20 (20:1), from about 5 (5:1) to about 15 (15:1), from about 5(5:1) to about 10 (10:1), or about 5 (5:1), 6 (6:1), 7 (7:1), 8 (8:1), 9(9:1), 10 (10:1), 11 (11:1), 12 (12:1), 13 (13:1), 14 (14:1), 15 (15:1),16 (16:1), 17 (17:1), 18 (18:1), 19 (19:1), 20 (20:1), 21 (21:1), 22(22:1), 23 (23:1), 24 (24:1), or 25 (25:1), or any fraction thereof orrange therein. The ratio of the starting materials (input) also fallswithin this range.

As previously discussed, the conjugated lipid may further include a CPL.A variety of general methods for making SNALP-CPLs (CPL-containingSNALP) are discussed herein. Two general techniques include the“post-insertion” technique, that is, insertion of a CPL into, forexample, a pre-formed SNALP, and the “standard” technique, wherein theCPL is included in the lipid mixture during, for example, the SNALPformation steps. The post-insertion technique results in SNALP havingCPLs mainly in the external face of the SNALP bilayer membrane, whereasstandard techniques provide SNALP having CPLs on both internal andexternal faces. The method is especially useful for vesicles made fromphospholipids (which can contain cholesterol) and also for vesiclescontaining PEG-lipids (such as PEG-DAAs and PEG-DAGs). Methods of makingSNALP-CPLs are taught, for example, in U.S. Pat. Nos. 5,705,385;6,586,410; 5,981,501; 6,534,484; and 6,852,334; U.S. Patent PublicationNo. 20020072121; and PCT Publication No. WO 00/62813, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes.

VII. Kits

The present invention also provides lipid particles (e.g., SNALP) in kitform. In some embodiments, the kit comprises a container which iscompartmentalized for holding the various elements of the lipidparticles (e.g., the active agents or therapeutic agents such as nucleicacids and the individual lipid components of the particles). Preferably,the kit comprises a container (e.g., a vial or ampoule) which holds thelipid particles of the invention (e.g., SNALP), wherein the particlesare produced by one of the processes set forth herein. In someembodiments, the kit may further comprise one or more antioxidants suchas metal chelators (e.g., EDTA), primary antioxidants, and/or secondaryantioxidants. In other embodiments, the kit may further comprise anendosomal membrane destabilizer (e.g., calcium ions). The kit typicallycontains the particle compositions of the present invention, either as asuspension in a pharmaceutically acceptable carrier or in dehydratedform, with instructions for their rehydration (if lyophilized) andadministration.

The lipid particles of the present invention can be tailored topreferentially target particular tissues, organs, or tumors of interest.In certain instances, preferential targeting of lipid particles such asSNALP may be carried out by controlling the composition of the particleitself. In some instances, the 1:57 lipid particle (e.g., SNALP)formulation can be used to preferentially target the liver (e.g., normalliver tissue). In other instances, the 7:54 lipid particle (e.g., SNALP)formulation can be used to preferentially target solid tumors such asliver tumors and tumors outside of the liver. In preferred embodiments,the kits of the invention comprise these liver-directed and/ortumor-directed lipid particles, wherein the particles are present in acontainer as a suspension or in dehydrated form.

In certain instances, it may be desirable to have a targeting moietyattached to the surface of the lipid particle to further enhance thetargeting of the particle. Methods of attaching targeting moieties(e.g., antibodies, proteins, etc.) to lipids (such as those used in thepresent particles) are known to those of skill in the art.

VIII. Administration of Lipid Particles

Once formed, the lipid particles of the invention (e.g., SNALP) areuseful for the introduction of active agents or therapeutic agents(e.g., nucleic acids such as interfering RNA) into cells. Accordingly,the present invention also provides methods for introducing an activeagent or therapeutic agent such as a nucleic acid (e.g., interferingRNA) into a cell. In some instances, the cell is a liver cell such as,e.g., a hepatocyte present in liver tissue. In other instances, the cellis a tumor cell such as, e.g., a tumor cell present in a solid tumor.The methods are carried out in vitro or in vivo by first forming theparticles as described above and then contacting the particles with thecells for a period of time sufficient for delivery of the active agentor therapeutic agent to the cells to occur.

The lipid particles of the invention (e.g., SNALP) can be adsorbed toalmost any cell type with which they are mixed or contacted. Onceadsorbed, the particles can either be endocytosed by a portion of thecells, exchange lipids with cell membranes, or fuse with the cells.Transfer or incorporation of the active agent or therapeutic agent(e.g., nucleic acid) portion of the particle can take place via any oneof these pathways. In particular, when fusion takes place, the particlemembrane is integrated into the cell membrane and the contents of theparticle combine with the intracellular fluid.

The lipid particles of the invention (e.g., SNALP) can be administeredeither alone or in a mixture with a pharmaceutically acceptable carrier(e.g., physiological saline or phosphate buffer) selected in accordancewith the route of administration and standard pharmaceutical practice.Generally, normal buffered saline (e.g., 135-150 mM NaCl) will beemployed as the pharmaceutically acceptable carrier. Other suitablecarriers include, e.g., water, buffered water, 0.4% saline, 0.3%glycine, and the like, including glycoproteins for enhanced stability,such as albumin, lipoprotein, globulin, etc. Additional suitablecarriers are described in, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES,Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985). As usedherein, “carrier” includes any and all solvents, dispersion media,vehicles, coatings, diluents, antibacterial and antifungal agents,isotonic and absorption delaying agents, buffers, carrier solutions,suspensions, colloids, and the like. The phrase “pharmaceuticallyacceptable” refers to molecular entities and compositions that do notproduce an allergic or similar untoward reaction when administered to ahuman.

The pharmaceutically acceptable carrier is generally added followinglipid particle formation. Thus, after the lipid particle (e.g., SNALP)is formed, the particle can be diluted into pharmaceutically acceptablecarriers such as normal buffered saline.

The concentration of particles in the pharmaceutical formulations canvary widely, i.e., from less than about 0.05%, usually at or at leastabout 2 to 5%, to as much as about 10 to 90% by weight, and will beselected primarily by fluid volumes, viscosities, etc., in accordancewith the particular mode of administration selected. For example, theconcentration may be increased to lower the fluid load associated withtreatment. This may be particularly desirable in patients havingatherosclerosis-associated congestive heart failure or severehypertension. Alternatively, particles composed of irritating lipids maybe diluted to low concentrations to lessen inflammation at the site ofadministration.

The pharmaceutical compositions of the present invention may besterilized by conventional, well-known sterilization techniques. Aqueoussolutions can be packaged for use or filtered under aseptic conditionsand lyophilized, the lyophilized preparation being combined with asterile aqueous solution prior to administration. The compositions cancontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, andcalcium chloride. Additionally, the particle suspension may includelipid-protective agents which protect lipids against free-radical andlipid-peroxidative damages on storage. Lipophilic free-radicalquenchers, such as alphatocopherol, and water-soluble iron-specificchelators, such as ferrioxamine, are suitable.

In some embodiments, the lipid particles of the invention (e.g., SNALP)are particularly useful in methods for the therapeutic delivery of oneor more nucleic acids comprising an interfering RNA sequence (e.g.,siRNA). In particular, it is an object of this invention to provide invitro and in vivo methods for treatment of a disease or disorder in amammal (e.g., a rodent such as a mouse or a primate such as a human,chimpanzee, or monkey) by downregulating or silencing the transcriptionand/or translation of one or more target nucleic acid sequences or genesof interest. As a non-limiting example, the methods of the invention areuseful for in vivo delivery of interfering RNA (e.g., siRNA) to theliver and/or tumor of a mammalian subject. In certain embodiments, thedisease or disorder is associated with expression and/or overexpressionof a gene and expression or overexpression of the gene is reduced by theinterfering RNA (e.g., siRNA). In certain other embodiments, atherapeutically effective amount of the lipid particle may beadministered to the mammal. In some instances, an interfering RNA (e.g.,siRNA) is formulated into a SNALP, and the particles are administered topatients requiring such treatment. In other instances, cells are removedfrom a patient, the interfering RNA is delivered in vitro (e.g., using aSNALP described herein), and the cells are reinjected into the patient.

A. In Vivo Administration

Systemic delivery for in vivo therapy, e.g., delivery of a therapeuticnucleic acid to a distal target cell via body systems such as thecirculation, has been achieved using nucleic acid-lipid particles suchas those described in PCT Publication Nos. WO 05/007196, WO 05/121348,WO 05/120152, and WO 04/002453, the disclosures of which are hereinincorporated by reference in their entirety for all purposes. Thepresent invention also provides fully encapsulated lipid particles thatprotect the nucleic acid from nuclease degradation in serum, arenon-immunogenic, are small in size, and are suitable for repeat dosing.

For in vivo administration, administration can be in any manner known inthe art, e.g., by injection, oral administration, inhalation (e.g.,intransal or intratracheal), transdermal application, or rectaladministration. Administration can be accomplished via single or divideddoses. The pharmaceutical compositions can be administered parenterally,i.e., intraarticularly, intravenously, intraperitoneally,subcutaneously, or intramuscularly. In some embodiments, thepharmaceutical compositions are administered intravenously orintraperitoneally by a bolus injection (see, e.g., U.S. Pat. No.5,286,634). Intracellular nucleic acid delivery has also been discussedin Straubringer et al., Methods Enzymol., 101:512 (1983); Mannino etal., Biotechniques, 6:682 (1988); Nicolau et al., Crit. Rev. Ther. DrugCarrier Syst., 6:239 (1989); and Behr, Acc. Chem. Res., 26:274 (1993).Still other methods of administering lipid-based therapeutics aredescribed in, for example, U.S. Pat. Nos. 3,993,754; 4,145,410;4,235,871; 4,224,179; 4,522,803; and 4,588,578. The lipid particles canbe administered by direct injection at the site of disease or byinjection at a site distal from the site of disease (see, e.g., Culver,HUMAN GENE THERAPY, MaryAnn Liebert, Inc., Publishers, New York. pp.70-71 (1994)). The disclosures of the above-described references areherein incorporated by reference in their entirety for all purposes.

In embodiments where the lipid particles of the present invention (e.g.,SNALP) are administered intravenously, at least about 5%, 10%, 15%, 20%,or 25% of the total injected dose of the particles is present in plasmaabout 8, 12, 24, 36, or 48 hours after injection. In other embodiments,more than about 20%, 30%, 40% and as much as about 60%, 70% or 80% ofthe total injected dose of the lipid particles is present in plasmaabout 8, 12, 24, 36, or 48 hours after injection. In certain instances,more than about 10% of a plurality of the particles is present in theplasma of a mammal about 1 hour after administration. In certain otherinstances, the presence of the lipid particles is detectable at leastabout 1 hour after administration of the particle. In certainembodiments, the presence of a therapeutic agent such as a nucleic acidis detectable in cells of the lung, liver, tumor, or at a site ofinflammation at about 8, 12, 24, 36, 48, 60, 72 or 96 hours afteradministration. In other embodiments; downregulation of expression of atarget sequence by an interfering RNA (e.g., siRNA) is detectable atabout 8, 12, 24, 36, 48, 60, 72 or 96 hours after administration. In yetother embodiments, downregulation of expression of a target sequence byan interfering RNA (e.g., siRNA) occurs preferentially in liver cells(e.g., hepatocytes), tumor cells, or in cells at a site of inflammation.In further embodiments, the presence or effect of an interfering RNA(e.g., siRNA) in cells at a site proximal or distal to the site ofadministration or in cells of the lung, liver, or a tumor is detectableat about 12, 24, 48, 72, or 96 hours, or at about 6, 8, 10, 12, 14, 16,18, 19, 20, 22, 24, 26, or 28 days after administration. In additionalembodiments, the lipid particles (e.g., SNALP) of the invention areadministered parenterally or intraperitoneally.

The compositions of the present invention, either alone or incombination with other suitable components, can be made into aerosolformulations (i.e., they can be “nebulized”) to be administered viainhalation (e.g., intranasally or intratracheally) (see, Brigham et al.,Am. J Sci., 298:278 (1989)). Aerosol formulations can be placed intopressurized acceptable propellants, such as dichlorodifluoromethane,propane, nitrogen, and the like.

In certain embodiments, the pharmaceutical compositions may be deliveredby intranasal sprays, inhalation, and/or other aerosol deliveryvehicles. Methods for delivering nucleic acid compositions directly tothe lungs via nasal aerosol sprays have been described, e.g., in U.S.Pat. Nos. 5,756,353 and 5,804,212. Likewise, the delivery of drugs usingintranasal microparticle resins and lysophosphatidyl-glycerol compounds(U.S. Pat. No. 5,725,871) are also well-known in the pharmaceuticalarts. Similarly, transmucosal drug delivery in the form of apolytetrafluoroetheylene support matrix is described in U.S. Pat. No.5,780,045. The disclosures of the above-described patents are hereinincorporated by reference in their entirety for all purposes.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In the practice of this invention, compositions are preferablyadministered, for example, by intravenous infusion, orally, topically,intraperitoneally, intravesically, or intrathecally.

Generally, when administered intravenously, the lipid particleformulations are formulated with a suitable pharmaceutical carrier. Manypharmaceutically acceptable carriers may be employed in the compositionsand methods of the present invention. Suitable formulations for use inthe present invention are found, for example, in REMINGTON'SPHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, Pa.,17th ed. (1985). A variety of aqueous carriers may be used, for example,water, buffered water, 0.4% saline, 0.3% glycine, and the like, and mayinclude glycoproteins for enhanced stability, such as albumin,lipoprotein, globulin, etc. Generally, normal buffered saline (135-150mM NaCl) will be employed as the pharmaceutically acceptable carrier,but other suitable carriers will suffice. These compositions can besterilized by conventional liposomal sterilization techniques, such asfiltration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamine oleate, etc. Thesecompositions can be sterilized using the techniques referred to aboveor, alternatively, they can be produced under sterile conditions. Theresulting aqueous solutions may be packaged for use or filtered underaseptic conditions and lyophilized, the lyophilized preparation beingcombined with a sterile aqueous solution prior to administration.

In certain applications, the lipid particles disclosed herein may bedelivered via oral administration to the individual. The particles maybe incorporated with excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, pills, lozenges, elixirs,mouthwash, suspensions, oral sprays, syrups, wafers, and the like (see,e.g., U.S. Pat. Nos. 5,641,515, 5,580,579, and 5,792,451, thedisclosures of which are herein incorporated by reference in theirentirety for all purposes). These oral dosage forms may also contain thefollowing: binders, gelatin; excipients, lubricants, and/or flavoringagents. When the unit dosage form is a capsule, it may contain, inaddition to the materials described above, a liquid carrier. Variousother materials may be present as coatings or to otherwise modify thephysical form of the dosage unit. Of course, any material used inpreparing any unit dosage form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed.

Typically, these oral formulations may contain at least about 0.1% ofthe lipid particles or more, although the percentage of the particlesmay, of course, be varied and may conveniently be between about 1% or 2%and about 60% or 70% or more of the weight or volume of the totalformulation. Naturally, the amount of particles in each therapeuticallyuseful composition may be prepared is such a way that a suitable dosagewill be obtained in any given unit dose of the compound. Factors such assolubility, bioavailability, biological half-life, route ofadministration, product shelf life, as well as other pharmacologicalconsiderations will be contemplated by one skilled in the art ofpreparing such pharmaceutical formulations, and as such, a variety ofdosages and treatment regimens may be desirable.

Formulations suitable for oral administration can consist of: (a) liquidsolutions, such as an effective amount of a packaged therapeutic agentsuch as nucleic acid (e.g., interfering RNA) suspended in diluents suchas water, saline, or PEG 400; (b) capsules, sachets, or tablets, eachcontaining a predetermined amount of a therapeutic agent such as nucleicacid (e.g., interfering RNA), as liquids, solids, granules, or gelatin;(c) suspensions in an appropriate liquid; and (d) suitable emulsions.Tablet forms can include one or more of lactose, sucrose, mannitol,sorbitol, calcium phosphates, corn starch, potato starch,microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc,magnesium stearate, stearic acid, and other excipients, colorants,fillers, binders, diluents, buffering agents, moistening agents,preservatives, flavoring agents, dyes, disintegrating agents, andpharmaceutically compatible carriers. Lozenge forms can comprise atherapeutic agent such as nucleic acid (e.g., interfering RNA) in aflavor, e.g., sucrose, as well as pastilles comprising the therapeuticagent in an inert base, such as gelatin and glycerin or sucrose andacacia emulsions, gels, and the like containing, in addition to thetherapeutic agent, carriers known in the art.

In another example of their use, lipid particles can be incorporatedinto a broad range of topical dosage forms. For instance, a suspensioncontaining nucleic acid-lipid particles such as SNALP can be formulatedand administered as gels, oils, emulsions, topical creams, pastes,ointments, lotions, foams, mousses, and the like.

When preparing pharmaceutical preparations of the lipid particles of theinvention, it is preferable to use quantities of the particles whichhave been purified to reduce or eliminate empty particles or particleswith therapeutic agents such as nucleic acid associated with theexternal surface.

The methods of the present invention may be practiced in a variety ofhosts. Preferred hosts include mammalian species, such as primates(e.g., humans and chimpanzees as well as other nonhuman primates),canines, felines, equines, bovines, ovines, caprines, rodents (e.g.,rats and mice), lagomorphs, and swine.

The amount of particles administered will depend upon the ratio oftherapeutic agent (e.g., nucleic acid) to lipid, the particulartherapeutic agent (e.g., nucleic acid) used, the disease or disorderbeing treated, the age, weight, and condition of the patient, and thejudgment of the clinician, but will generally be between about 0.01 andabout 50 mg per kilogram of body weight, preferably between about 0.1and about 5 mg/kg of body weight, or about 10⁸-10¹⁰ particles peradministration (e.g., injection).

B. In Vitro Administration

For in vitro applications, the delivery of therapeutic agents such asnucleic acids (e.g., interfering RNA) can be to any cell grown inculture, whether of plant or animal origin, vertebrate or invertebrate,and of any tissue or type. In preferred embodiments, the cells areanimal cells, more preferably mammalian cells, and most preferably humancells (e.g., tumor cells or hepatocytes).

Contact between the cells and the lipid particles, when carried out invitro, takes place in a biologically compatible medium. Theconcentration of particles varies widely depending on the particularapplication, but is generally between about 1 mol and about 10 mmol.Treatment of the cells with the lipid particles is generally carried outat physiological temperatures (about 37° C.) for periods of time of fromabout 1 to 48 hours, preferably of from about 2 to 4 hours.

In one group of preferred embodiments, a lipid particle suspension isadded to 60-80% confluent plated cells having a cell density of fromabout 10³ to about 10⁵ cells/ml, more preferably about 2×10⁴ cells/ml.The concentration of the suspension added to the cells is preferably offrom about 0.01 to 0.2 μg/ml, more preferably about 0.1 gig/ml.

To the extent that tissue culture of cells may be required, it iswell-known in the art. For example, Freshney, Culture of Animal Cells, aManual of Basic Technique, 3rd Ed., Wiley-Liss, New York (1994), Kuchleret al., Biochemical Methods in Cell Culture and Virology, Dowden,Hutchinson and Ross, Inc. (1977), and the references cited thereinprovide a general guide to the culture of cells. Cultured cell systemsoften will be in the form of monolayers of cells, although cellsuspensions are also used.

Using an Endosomal Release Parameter (ERP) assay, the deliveryefficiency of the SNALP or other lipid particle of the invention can beoptimized. An ERP assay is described in detail in U.S. PatentPublication No. 20030077829, the disclosure of which is hereinincorporated by reference in its entirety for all purposes. Moreparticularly, the purpose of an ERP assay is to distinguish the effectof various cationic lipids and helper lipid components of SNALP or otherlipid particle based on their relative effect on binding/uptake orfusion with/destabilization of the endosomal membrane. This assay allowsone to determine quantitatively how each component of the SNALP or otherlipid particle affects delivery efficiency, thereby optimizing the SNALPor other lipid particle. Usually, an ERP assay measures expression of areporter protein (e.g., luciferase, β-galactosidase, green fluorescentprotein (GFP), etc.), and in some instances, a SNALP formulationoptimized for an expression plasmid will also be appropriate forencapsulating an interfering RNA. In other instances, an ERP assay canbe adapted to measure downregulation of transcription or translation ofa target sequence in the presence or absence of an interfering RNA(e.g., siRNA). By comparing the ERPs for each of the various SNALP orother lipid particles, one can readily determine the optimized system,e.g., the SNALP or other lipid particle that has the greatest uptake inthe cell.

C. Cells for Delivery of Lipid Particles

The compositions and methods of the present invention are used to treata wide variety of cell types, in vivo and in vitro. Suitable cellsinclude, but are not limited to, hepatocytes, reticuloendothelial cells(e.g., monocytes, macrophages, etc.), fibroblast cells, endothelialcells, platelet cells, other cell types infected and/or susceptible ofbeing infected with viruses, hematopoietic precursor (stem) cells,keratinocytes, skeletal and smooth muscle cells, osteoblasts, neurons,quiescent lymphocytes, terminally differentiated cells, slow ornoncycling primary cells, parenchymal cells, lymphoid cells, epithelialcells, bone cells, and the like.

In particular embodiments, an active agent or therapeutic agent such asa nucleic acid (e.g., an interfering RNA) is delivered to cancer cells(e.g., cells of a solid tumor) including, but not limited to, livercancer cells, lung cancer cells, colon cancer cells, rectal cancercells, anal cancer cells, bile duct cancer cells, small intestine cancercells, stomach (gastric) cancer cells, esophageal cancer cells,gallbladder cancer cells, pancreatic cancer cells, appendix cancercells, breast cancer cells, ovarian cancer cells, cervical cancer cells,prostate cancer cells, renal cancer cells, cancer cells of the centralnervous system, glioblastoma tumor cells, skin cancer cells, lymphomacells, choriocarcinoma tumor cells, head and neck cancer cells,osteogenic sarcoma tumor cells, and blood cancer cells.

In vivo delivery of lipid particles such as SNALP encapsulating anucleic acid (e.g., an interfering RNA) is suited for targeting cells ofany cell type. The methods and compositions can be employed with cellsof a wide variety of vertebrates, including mammals, such as, e.g,canines, felines, equines, bovines, ovines, caprines, rodents (e.g.,mice, rats, and guinea pigs), lagomorphs, swine, and primates (e.g.monkeys, chimpanzees, and humans).

D. Detection of Lipid Particles

In some embodiments, the lipid particles of the present invention (e.g.,SNALP) are detectable in the subject at about 1, 2, 3, 4, 5, 6, 7, 8 ormore hours. In other embodiments, the lipid particles of the presentinvention (e.g., SNALP) are detectable in the subject at about 8, 12,24, 48, 60, 72, or 96 hours, or about 6, 8, 10, 12, 14, 16, 18, 19, 22,24, 25, or 28 days after administration of the particles. The presenceof the particles can be detected in the cells, tissues, or otherbiological samples from the subject. The particles may be detected,e.g., by direct detection of the particles, detection of a therapeuticnucleic acid such as an interfering RNA (e.g., siRNA) sequence,detection of the target sequence of interest (i.e., by detectingexpression or reduced expression of the sequence of interest), or acombination thereof.

1. Detection of Particles

Lipid particles of the invention such as SNALP can be detected using anymethod known in the art. For example, a label can be coupled directly orindirectly to a component of the lipid particle using methods well-knownin the art. A wide variety of labels can be used, with the choice oflabel depending on sensitivity required, ease of conjugation with thelipid particle component, stability requirements, and availableinstrumentation and disposal provisions. Suitable labels include, butare not limited to, spectral labels such as fluorescent dyes (e.g.,fluorescein and derivatives, such as fluorescein isothiocyanate (FITC)and Oregon Green™; rhodamine and derivatives such Texas red,tetrarhodimine isothiocynate (TRITC), etc., digoxigenin, biotin,phycoerythrin, AMCA, CyDyes™, and the like; radiolabels such as ³H,¹²⁵I, ³⁵S, ¹⁴C, ³²P, ³³P, etc.; enzymes such as horse radish peroxidase,alkaline phosphatase, etc.; spectral colorimetric labels such ascolloidal gold or colored glass or plastic beads such as polystyrene,polypropylene, latex, etc. The label can be detected using any meansknown in the art.

2. Detection of Nucleic Acids

Nucleic acids (e.g., interfering RNA) are detected and quantified hereinby any of a number of means well-known to those of skill in the art. Thedetection of nucleic acids may proceed by well-known methods such asSouthern analysis, Northern analysis, gel electrophoresis, PCR,radiolabeling, scintillation counting, and affinity chromatography.Additional analytic biochemical methods such as spectrophotometry,radiography, electrophoresis, capillary electrophoresis, highperformance liquid chromatography (HPLC), thin layer chromatography(TLC), and hyperdiffusion chromatography may also be employed.

The selection of a nucleic acid hybridization format is not critical. Avariety of nucleic acid hybridization formats are known to those skilledin the art. For example, common formats include sandwich assays andcompetition or displacement assays. Hybridization techniques aregenerally described in, e.g., “Nucleic Acid Hybridization, A PracticalApproach,” Eds. Hames and Higgins, IRL Press (1985).

The sensitivity of the hybridization assays may be enhanced through theuse of a nucleic acid amplification system which multiplies the targetnucleic acid being detected. In vitro amplification techniques suitablefor amplifying sequences for use as molecular probes or for generatingnucleic acid fragments for subsequent subcloning are known. Examples oftechniques sufficient to direct persons of skill through such in vitroamplification methods, including the polymerase chain reaction (PCR),the ligase chain reaction (LCR), QPβ-replicase amplification, and otherRNA polymerase mediated techniques (e.g., NASBA™) are found in Sambrooket al., In Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press (2000); and Ausubel et al., SHORT PROTOCOLS INMOLECULAR BIOLOGY, eds., Current Protocols, Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc. (2002); as well as U.S.Pat. No. 4,683,202; PCR Protocols, A Guide to Methods and Applications(Innis et al. eds.) Academic Press Inc. San Diego, Calif. (1990);Arnheim & Levinson (Oct. 1, 1990), C&EN 36; The Journal Of NIH Research,3:81 (1991); Kwoh et al., Proc. Natl. Acad. Sci. USA, 86:1173 (1989);Guatelli et al., Proc. Natl. Acad. Sci. USA, 87:1874 (1990); Lomell etal., J. Clin. Chem., 35:1826 (1989); Landegren et al., Science, 241:1077(1988); Van Brunt, Biotechnology, 8:291 (1990); Wu and Wallace, Gene,4:560 (1989); Barringer et al., Gene, 89:117 (1990); and Sooknanan andMalek, Biotechnology, 13:563 (1995). Improved methods of cloning invitro amplified nucleic acids are described in U.S. Pat. No. 5,426,039.Other methods described in the art are the nucleic acid sequence basedamplification (NASBA™, Cangene, Mississauga, Ontario) and Qβ-replicasesystems. These systems can be used to directly identify mutants wherethe PCR or LCR primers are designed to be extended or ligated only whena select sequence is present. Alternatively, the select sequences can begenerally amplified using, for example, nonspecific PCR primers and theamplified target region later probed for a specific sequence indicativeof a mutation. The disclosures of the above-described references areherein incorporated by reference in their entirety for all purposes.

Nucleic acids for use as probes, e.g., in in vitro amplificationmethods, for use as gene probes, or as inhibitor components aretypically synthesized chemically according to the solid phasephosphoramidite triester method described by Beaucage et al.,Tetrahedron Letts., 22:1859 1862 (1981), e.g., using an automatedsynthesizer, as described in Needham VanDevanter et al., Nucleic AcidsRes., 12:6159 (1984). Purification of polynucleotides, where necessary,is typically performed by either native acrylamide gel electrophoresisor by anion exchange HPLC as described in Pearson et al., J. Chrom.,255:137 149 (1983). The sequence of the synthetic polynucleotides can beverified using the chemical degradation method of Maxam and Gilbert(1980) in Grossman and Moldave (eds.) Academic Press, New York, Methodsin Enzymology, 65:499.

An alternative means for determining the level of transcription is insitu hybridization. In situ hybridization assays are well-known and aregenerally described in Angerer et al., Methods Enzymol., 152:649 (1987).In an in situ hybridization assay, cells are fixed to a solid support,typically a glass slide. If DNA is to be probed, the cells are denaturedwith heat or alkali. The cells are then contacted with a hybridizationsolution at a moderate temperature to permit annealing of specificprobes that are labeled. The probes are preferably labeled withradioisotopes or fluorescent reporters.

IX. Examples

The present invention will be described in greater detail by way ofspecific examples. The following examples are offered for illustrativepurposes, and are not intended to limit the invention in any manner.Those of skill in the art will readily recognize a variety ofnoncritical parameters which can be changed or modified to yieldessentially the same results.

Example 1. Synthesis of CP-DODMA

CP-DODMA (Compound 1) having the structure shown below was synthesizedas described below.

A solution of DODMA (310 mg, 0.5 mmol) in anhydrous dichloromethane (10mL) under nitrogen was cooled to 0° C. and a 1M solution of diethylzincin hexanes (5 mL, 5 mmol, 5 eqv) added. The solution was stirred for 1hour at 0° C. then diiodomethane (1.34 g, 404 μL, 5 mmol) was added andthe solution was stirred for 16 hours at room temperature undernitrogen. TLC (8% MeOH in CHCl₃) showed that the starting material wasconsumed and a very slightly more polar product had formed. The reactionmixture was concentrated and purified by column chromatography. A polar(lower running) impurity coeluted with the product. After concentratingappropriate column fractions they were dissolved in EtOAc and washedwith 5% HCl (2×10 mL), water (10 mL), sat. NaHCO₃ (10 mL), water (10 mL)and brine (10 mL). The solution was dried over MgSO₄ and concentrated toafford a clear pale yellow oil (200 mg, 62%). ¹H NMR analysis showedcomplete conversion of the cis-alkenes to cyclopropyl groups.

Example 2. Synthesis of CP-DPetroDMA

CP-DPetroDMA (Compound 2) having the structure shown below wassynthesized as described below.

A solution of DPetroDMA (300 mg, 0.48 mmol) in anhydrous dichloromethane(10 mL) under nitrogen was cooled to 0° C. and a 1M solution ofdiethylzinc in hexanes (5 mL, 5 mmol, 5 eqv) added. The solution wasstirred for 1 hour at 0° C. then diiodomethane (1.34 g, 404 μL, 5 mmol)was added and the solution was stirred overnight at room temperatureunder nitrogen. TLC (8% MeOH in CHCl₃) showed that the starting materialwas consumed and a very slightly more polar product had formed. Thereaction mixture was concentrated and purified by column chromatography.A polar (lower running) impurity coeluted with the product. Afterconcentrating appropriate column fractions they were dissolved in EtOAcand washed with 5% HCl (2×10 mL), water (10 mL), sat. NaHCO₃ (10 mL),water (10 mL) and brine (10 mL). The solution was dried over MgSO₄ andconcentrated to afford a clear pale yellow oil (250 mg, 0.39 mmol, 80%).¹H NMR analysis showed complete conversion of the cis-alkenes tocyclopropyl groups.

Example 3. Synthesis of CP-DLinDMA

CP-DLinDMA (Compound 3) having the structure shown below was synthesizedas described in Scheme 1 below.

Reagent MW Amount mmol Equivalents 1 DLinDMA 615.06 300 mg 0.49 1 2Dichloromethane — 20 mL — — 3 Diethylzinc 1M in hexanes — 4.9 mL 4.9 104 Diiodomethane 267.84 2.62 g 9.8 20 (790 μL)

To a solution of DLinDMA (300 mg, 0.49 mmol) in anhydrousdichloromethane (20 mL) cooled to 0° C. under nitrogen was added a 1Msolution of diethylzinc in hexanes (4.9 mL). The solution was stirredfor 1 hour at 0° C. then diiodomethane (2.62 g, 9.8 mmol) was added andthe solution was stirred for 16 hours at room temperature undernitrogen. The solution was diluted with dichloromethane (20 mL),filtered and concentrated in vacuo to dryness. The residue was purifiedby column chromatography (10″ L×0.5″ D; eluted with 2.5% MeOH in CHC₃)to afford the product as a light yellow oil (288 mg, 87%). See also,Tanaka et al., Bioorg. Med. Chem. Lett., 13:1037-1040 (2003).

Example 4. Synthesis of CP-DLenDMA

CP-DLenDMA (Compound 4) having the structure shown below was synthesizedas described in Scheme 2 below.

Reagent MW Amount mmol Equivalents 1 DLenDMA 611.02 67 mg 0.11 1 2Dichloromethane — 5 mL — — 3 Diethylzinc 1M in hexanes — 1.48 mL 1.48 144 Diiodomethane 267.84 790 mg 2.95 27 (237 μL)

To a solution of DLenDMA (67 mg, 0.11 mmol) in anhydrous dichloromethane(5 mL) cooled to 0° C. under nitrogen was added a 1M solution ofdiethylzinc in hexanes (1.48 mL). The solution was stirred for 1 hour at0° C. then diiodomethane (790 mg, 2.95 mmol) was added and the solutionwas stirred for 16 hours at room temperature under nitrogen. TLC (8%MeOH in CHCl₃) showed that the starting material was consumed and a veryslightly more polar product had formed. The solution was purified bycolumn chromatography without a workup (0.5″ D×8″ L; eluted with 4% MeOHin CHCl₃) to afford a clear pale yellow oil (73 mg, 96%). ¹H NMRanalysis showed complete conversion of the cis-alkenes to cyclopropyl.See also, Tanaka et al., Bioorg. Med. Chem. Lett., 13:1037-1040 (2003).

Example 5. Synthesis of γ-DLenDMA

γ-DLenDMA (Compound 5) having the structure shown below was synthesizedas described below.

A 250 mL round bottom flask was charged with3-(dimethylamino)-1,2-propanediol (0.8 g, 6.7 mmol), tetrabutylammoniumhydrogen sulphate (1 g), gamma linolenyl mesylate(cis-6,9,12-octadecatriene sulphonic acid) (5 g, 14.6 mmol), and 30 mLtoluene. After stirring for 15 minutes, the reaction was cooled to 0-5°C. A solution of 40% sodium hydroxide (15 mL) was added slowly. Thereaction was left to stir for approximately 48 hours. An additional 15mL of toluene was then added to the reaction vessel, along with 40%sodium hydroxide (15 mL). After the reaction was stirred for anadditional 12 hours, water (50 mL) and isopropyl acetate (50 mL) wereadded and stirred for 15 minutes. The mixture was then transferred to a500 mL separatory funnel and allowed to separate. The lower aqueousphase was run off and the organic phase was washed with saturated sodiumchloride (2×50 mL). Since the aqueous and organic phases resulting fromthe saturated sodium chloride washes could not be completely separatedafter 20 minutes, the lower aqueous phase (slightly yellow) was run offand back extracted with chloroform (˜45 mL). The organic phase was driedwith MgSO₄, filtered, and the solvent evaporated.

The crude product, an orange liquid, was purified on columnchromatography using silica gel (60 g) with 0-3% methanol gradient indichloromethane to yield 3.19 g. The product was further purified viacolumn chromatography on silica gel (50 g) with 10-30% ethyl acetategradient in hexanes to yield 1.26 g pure product.

Example 6. Synthesis of CP-γ-DLenDMA

CP-γ-DLenDMA (Compound 6) having the structure shown below wassynthesized as described in Scheme 3 below.

Reagent MW Amount mmol Equivalents 1 γ-DLenDMA 612.02 100 mg 0.16 1 2Dichloromethane — 10 mL — — 3 Diethylzinc 1M in hexanes — 2.5 mL 2.45 154 Diiodomethane 267.84 1.29 g 4.8 30 (400 μL)

To a solution of γ-DLenDMA (Compound 5) (100 mg, 0.16 mmol) in anhydrousdichloromethane (10 mL) cooled to 0° C. under nitrogen was added a IMsolution of diethylzinc in hexanes (2.5 mL, 2.45 mmol). The solution wasstirred for 1 hour at 0° C. then diiodomethane (1.29 g, 4.8 mmol) wasadded and the solution was stirred for 16 hours at room temperatureunder nitrogen. Upon completion by TLC (8% MeOH in CHCl₃), the solutionwas concentrated in vacuo to dryness. The residue was purified by columnchromatography (10″ L×0.5″ D; eluted with 100% CHCl₃) to afford theproduct as a yellow oil (111 mg, 98%). See also, Tanaka et al., Bioorg.Med. Chem. Lett., 13:1037-1040 (2003).

Example 7. Synthesis of CP-DLen-C2K-DMA

CP-DLen-C2K-DMA (Compound 11) having the structure shown below wassynthesized as described in Scheme 4 below. CP-DLen-C2K-DMA is alsoknown as CP-linolenyl-C2K, CP-Len-C2K, and CP-DLen-C2K.

Synthesis of Dilinolenyl Ketone (Compound 7):

To a 1000 mL RBF containing a solution of dilinolenyl methanol (6.0 g,11.4 mmol) in anh. DCM (200 mL) was added pyridinium chlorochromate(7.39 g, 34.2 mmol), anh. sodium carbonate (1.0 g, 5.66 mmol) and astirbar. The resulting suspension was stirred under nitrogen at RT for 3h, after which time TLC indicated all SM to have been consumed. Ether(300 mL) was then added to the mixture and the resulting brownsuspension filtered through a pad of silica (300 mL), washing the padwith ether (3×100 mL). The ether phases were combined, concentrated andpurified to yield 4.2 g (8.0 mmol, 70%) of the ketone.

Synthesis of Linolenyl Ketal (Compound 8):

A 100 mL RBF was charged with dilinolenyl ketone (Compound 7) (4.2 g,8.2 mmol), 1,2,4-butanetriol (3.4 g, 32 mmol), PPTS (200 mg, 0.8 mmol)and a stir bar. The flask was flushed with nitrogen and anhydroustoluene (60 mL) added. The reaction vessel was fitted with a Dean Starktube and condenser and brought to reflux and the reaction was leftovernight. After cooling to room temperature, the reaction mixturediluted with toluene (50 mL), and washed with 5% aq. Na₂CO₃ (2×50 mL),water (50 mL), dried (MgSO₄) and purified by chromatography to yield 3.0g (4.9 mmol, 59%) of the ketal.

Mesylate Formation (Compound 9):

A 250 mL RBF was charged with the ketal (Compound 8) (3.0 g, 4.9 mmol),TEA (2.2 mL, 15.6 mmol) and a stir bar. The flask was flushed withnitrogen, anh. DCM (20 mL) added, and the solution cooled to −15° C. Ina separate 50 mL flask, a solution of MsCl (9.7 mmol, 2 eqv.) inanhydrous DCM (30 mL) was prepared, then transferred to the reactionvessel by syringe over 20 minutes. The reaction was stirred for 90minutes at −15° C., at which point starting material had been consumed.The reaction mixture was diluted with a further 50 mL of DCM, washedwith NaHCO₃ (2×50 mL), dried (MgSO₄) and purified by chromatography.Final yield 3.1 g, 4.5 mmol, 92%.

Synthesis of D-Len-C2K-DMA (Compound 10):

A 250 mL RBF was charged with the mesylate (Compound 9) (3.0 g, 4.35mmol), isopropanol (25 mL) and a stir bar. The flask was flushed withnitrogen, sealed, and a 2.0 M solution of dimethylamine in methanol (120mL) added via canulla. The reaction was stirred at room temperature for3 days. The solution was concentrated and purified by chromatography.Final yield 2.49 g, 3.9 mmol, 90%.

Synthesis of CP-DLen-C2K-DMA (Compound 11):

To a 250 mL RBF was added DLen-C2K-DMA (Compound 10) (1.28 g, 2 mmol), astirbar and anh. DCM (40 mL). The flask was flushed with N₂ and cooledto 0° C., then a 1M solution of diethylzinc in hexanes added (60 mL, 60mmol, 5 equivalents per olefin). The solution was stirred for 1 hour at0° C., then diiodomethane (4.84 mL 60 mmol). The reaction mixture wasconcentrated and then redissolved in EtOAc (50 mL). The EtOAc was washedsuccessively with 5% HCl (2×50 mL), water (50 mL), NaHCO₃ (50 mL), water(50 mL), and brine (50 mL). The aqueous washes were combined andextracted with DCM (2×50 mL). All organics were combined, dried andconcentrated to yield crude CP-Len-C2K. ¹H-NMR and HPLC indicated someolefins still to be present, so the compound was treated again, usingthe same procedures and amounts outlined above. This time ¹H-NMRindicated total conversion of the olefins. Final yield afterchromatography was 1.2 g, 1.66 mmol, 83%.

Example 8. Synthesis of CP-γDLen-C2K-DMA

CP-γDLen-C2K-DMA (Compound 16) having the structure shown below wassynthesized as described in Scheme 5 below. CP-γDLen-C2K-DMA is alsoknown as CP-γlinolenyl-C2K, CP-γLen-C2K, and CP-D-γ-Len-C2K.

Synthesis of Di-γ-Linolenyl Ketone (Compound 12):

To a 1000 mL RBF containing a solution of di-γ-linolenyl methanol (6.0g, 11.4 mmol) in anh. DCM (200 mL) was added pyridinium chlorochromate(7.39 g, 34.2 mmol), anh. sodium carbonate (1.0 g, 5.66 mmol) and astirbar. The resulting suspension was stirred under nitrogen at RT for 3h, after which time TLC indicated all SM to have been consumed. Ether(300 mL) was then added to the mixture and the resulting brownsuspension filtered through a pad of silica (300 mL), washing the padwith ether (3×100 mL). The ether phases were combined, concentrated andpurified to yield 5.5 g (10.5 mmol, 92%) of ketone.

Synthesis of γ-Linolenyl Ketal (Compound 13):

A 100 mL RBF was charged with di-γ-linolenyl ketone (Compound 12) (2.14g, 4.1 mmol), 1,2,4-butanetriol (1.7 g, 16.0 mmol), PPTS (100 mg, 0.4mmol) and a stir bar. The flask was flushed with nitrogen and anhydroustoluene (30 mL) added. The reaction vessel was fitted with a Dean Starktube and condenser and brought to reflux and the reaction was leftovernight. After cooling to room temperature, the reaction mixture waswashed with 5% aq. Na₂CO₃ (2×50 mL), water (50 mL), dried (MgSO₄) andpurified by chromatography to yield 1.34 g (2.2 mmol, 53%) of the ketal.

Mesylate Formation (Compound 14):

A 250 mL RBF was charged with the ketal (Compound 13) (1.34 g, 2.19mmol), TEA (1 mL, 7.1 mmol) and a stir bar. The flask was flushed withnitrogen, anh. DCM (10 mL) added, and the solution cooled to −15° C. Ina separate 50 mL flask, a solution of MsCl (342 μL, 4.4 mmol, 2 eqv.) inanhydrous DCM (15 mL) was prepared, then transferred to the reactionvessel by syringe over 20 minutes. The reaction was stirred for 90minutes at −15° C., at which point starting material had been consumed.The reaction mixture was diluted with a further 50 mL of DCM, washedwith NaHCO₃ (2×50 mL), dried (MgSO₄) and purified by chromatography.Final yield 1.31 g, 1.90 mmol, 87%.

Synthesis of D-γ-Len-C2K-DMA (Compound 15):

A 250 mL RBF was charged with the mesylate (Compound 14) (1.31 g, 1.9mmol), isopropanol (10 mL) and a stir bar. The flask was flushed withnitrogen, sealed, and a 2.0 M solution of dimethylamine in methanol (60mL) added via canulla. The reaction was stirred at room temperature for3 days. The solution was concentrated and purified by chromatography.Final yield 1.1 g, 1.72 mmol, 91%.

Synthesis of CP-γDLen-C2K-DMA (Compound 16):

To a 250 mL RBF was added γ-Len-C2K (Compound 15) (638 mg, 1 mmol), astirbar and anh. DCM (40 mL). The flask was flushed with N₂ and cooledto 0° C., then a 1M solution of diethylzinc in hexanes added (30 mL, 30mmol, 5 equivalents per olefin). The solution was stirred for 1 hour at0° C., then diiodomethane (2.42 mL 30 mmol). The reaction mixture wasconcentrated and then redissolved in EtOAc (50 mL). The EtOAc was washedsuccessively with 5% HCl (2×50 mL), water (50 mL), NaHCO₃ (50 mL), water(50 mL), and brine (50 mL). The aqueous washes were combined andextracted with DCM (2×50 mL). All organics were combined, dried andconcentrated to yield crude CP-γ-Len-C2K. As previously, ¹H-NMR and HPLCindicated some olefins still to be present, so the compound was treatedagain, using the same procedures and amounts outlined above. This time¹H-NMR indicated total conversion of the olefins. Final yield afterchromatography was 614 mg, 0.85 mmol, 85%.

Example 9. Synthesis of CP-C2K-DMA

CP-C2K-DMA (Compound 17) having the structure shown below wassynthesized as described in Scheme 6 below. CP-C2K-DMA is also known asCP-C2K.

To a solution of DLin-C2K-DMA (1.2 g, 1.87 mmol) in anhydrous CH₂Cl₂ (36mL) at 0° C. under nitrogen was added 2.5 equivalents diethyl zinc (1Msolution in hexanes) (18.7 mL) per olefin. The solution was stirred for75 minutes and then 2.5 equivalents diiodomethane (1.5 mL, 18.7 mmol)per olefin was added. The reaction was stirred overnight at room temp.The white suspension was poured into ice (100 mL) and diluted to 150 mLusing ethyl acetate (white solid dissolved upon the addition of ethylacetate). 5% HCl (50 mL) was added and the aqueous layer backextractedwith ethyl acetate (2×100 mL). The combined organics were washed with 5%HCl again, then saturated NaHCO₃, water, and brine (150 mL each), driedover MgSO₄, filtered, and concentrated to yield a brown yellow oil.

The above procedure was repeated once in order to ensure 100%cyclopropylation of the double bonds. The brown yellow oil was analyzedby HPLC and determined to be >99% pure. The oil was decolorized using asecond column (column 2″L×2″W; eluted with 10% ethyl acetate in hexanes)to afford the product as a pale yellow oil. Final yield 740 mg.

Example 10. Synthesis of LenMC3 and CP-LenMC3

LenMC3 (Compound 20) and CP-LenMC3 (Compound 21) having the structuresshown below were synthesized as described in Scheme 7 below. LenMC3 isalso known as linolenyl-MC3 and DLen-MC3. CP-LenMC3 is also known asCP-linolenyl-MC3 and CP-DLen-MC3.

Synthesis of Linolenyl Bromide (Compound 18)

Magnesium bromide etherate (34 g, 110 mmol) and a stir bar were added toa 2000 mL round bottom flask. The flask was sealed and flushed withnitrogen. Anhydrous diethyl ether (400 mL) was added via canulla. Asolution of linolenyl mesylate (20 g, 58 mmol) in anhydrous ether (300mL) was then added, and the suspension stirred overnight. The suspensionwas poured into 500 mL of chilled water and transferred to a 2000-mLseparating funnel. After shaking, the organic phase was separated. Theaqueous phase was then extracted with ether (2×250 mL) and all etherphases combined. The ether phase was washed with water (2×250 mL), brine(250 mL) and dried over anhydrous Mg₂SO₄. The solution was filtered,concentrated and purified by flash chromatography. Final yield 19.1 g,100%.

Synthesis of Dilinolenyl Methanol (Compound 19)

Magnesium turnings (2.1 g, 87 mmol), 5 crystals of iodine and a stirbarwere added to a 1000 mL round-bottom flask. The flask was flushed withnitrogen and a solution of linolenyl bromide (Compound 18) (19.1 g, 58mmol) in anhydrous diethyl ether (500 mL) added via cannula. The mixtureturned cloudy and was refluxed overnight. The mixture was cooled to RTand ethyl formate (4.66 mL, 58 mmol) added via syringe. The addition wasmade dropwise, directly into the reaction mixture, and the cloudysuspension again stirred overnight. During this time the reaction turnedbright yellow. The R.M. was transferred to a 2000-mL sep. funnel withether (50 mL), and washed with 10% H₂SO₄ (200 mL), water (2×200 mL) andbrine (200 mL). The organic was dried over anhydrous MgSO₄, filtered andconcentrated. Crude yield was 14.5 g. TLC indicated that majority ofproduct was the methyl formate, which was purified by columnchromatography. The purified formate (9.3 g, 16.7 mmol) was transferredto a 1000 mL round bottom flask and EtOH (600 mL) and a stirbar added.With stirring, water (25 mL-forming ˜5% aqueous solution) was slowlyadded, followed by KOH (2.0 g, 35.7 mmol). After 1 hour, the solutionhad turned pale yellow. TLC indicated reaction had gone to completion.The solution was concentrated by rotovap to 50% of its volume and thenpoured into 200 mL of 5% HCl. The aqueous phase was extracted with ether(3×200 mL). The ether fractions were combined and washed with water(3×200 mL), dried (MgSO₄) and concentrated to yield 8.9 g of dilinolenylmethanol (16.8 mmol, 58%).

Synthesis of Len-MC3 (Compound 20)

Dilinolenyl methanol (Compound 19) (2.5 g, 4.76 mmol),dimethylaminobutyric acid hydrochloride (970 mg, 5.77 mmol) and a stirbar were added to 100 mL RBF. The flask was flushed with nitrogen andanhydrous DCM (40 mL) added, followed by EDCI (FW 191.7, 1.2 g, 6.26mmol), DIPEA (2.1 mL, 12.1 mmol) and DMAP (150 mg, 1.23 mmol). Thereaction was stirred overnight, whereupon TLC indicated >80% conversion.Reaction was diluted with DCM (100 mL) and washed with sat. NaHCO₃ (100mL), water (200 mL) and sat. NaCL (100 mL). Aqueous washes were combinedand extracted with DCM (2×50 mL). Organics were then combined, dried(MgSO₄) and concentrated to yield a yellow oil with some crystallinematter. This was purified by chromatography to yield Len-MC3 as a paleyellow oil (2.3 g, 3.6 mmol, 76%).

Synthesis of CP-LenMC3 (Compound 21)

To a 250 mL RBF was added Len-MC3 (Compound 20) (1.1 g, 1.72 mmol), astirbar and anhydrous DCM (40 mL). The flask was flushed with N₂ andcooled to 0° C., then a IM solution of diethylzinc in hexanes added (30mL, 30 mmol). The solution was stirred for 1 hour at 0° C., thendiiodomethane (2.4 mL 30 mmol) added and the reaction stirred overnightat RT. The reaction mixture was concentrated and then redissolved inEtOAc (50 mL). The EtOAc was washed successively with 5% HCl (2×50 mL),water (50 mL), NaHCO₃ (50 mL), water (50 mL), and brine (50 mL). Theaqueous washes were combined and extracted with DCM (2×50 mL). Allorganics were combined, dried and concentrated to yield crudeCP-Len-MC3. ¹H-NMR indicated some olefins still to be present, so thecompound was treated again, using the same procedures and amountsoutlined above. This time, after chromatography, ¹H-NMR indicated totalconversion of the olefins. Final yield 1.0 g, 1.39 mmol, 81%.

Example 11. Synthesis of γ-LenMC3 and CP-γ-LenMC3

γ-LenMC3 (Compound 24) and CP-γ-LenMC3 (Compound 25) having thestructures shown below were synthesized as described in Scheme 8 below.γ-LenMC3 is also known as γlinolenyl-MC3, γDLen-MC3, and D-γ-Len-MC3.CP-γ-LenMC3 is also known as CP-γlinolenyl-MC3, CP-γDLen-MC3, andCP-D-γ-Len-MC3.

Synthesis of γ-Linolenyl Bromide (Compound 22)

Magnesium bromide etherate (34 g, 110 mmol) and a stir bar were added toa 2000 mL round bottom flask. The flask was sealed and flushed withnitrogen. Anhydrous diethyl ether (400 mL) was added via canulla. Asolution of γ-linolenyl mesylate (20 g, 58 mmol) in anhydrous ether (300mL) was then added, and the suspension stirred overnight. The suspensionwas poured into 500 mL of chilled water and transferred to a 2000-mLseparating funnel. After shaking, the organic phase was separated. Theaqueous phase was then extracted with ether (2×250 mL) and all etherphases combined. The ether phase was washed with water (2×250 mL), brine(250 mL) and dried over anhydrous Mg₂SO₄. The solution was filtered,concentrated and purified by flash chromatography. Final yield 18.9 g,99%.

Synthesis of Di-γ-Linolenyl Methanol (Compound 23)

Magnesium turnings (2.1 g, 87 mmol), 5 crystals of iodine and a stirbarwere added to a 1000 mL round-bottom flask. The flask was flushed withnitrogen and a solution of γ-linolenyl bromide (Compound 22) (18.9 g, 57mmol) in anhydrous diethyl ether (500 mL) added via cannula. The mixtureturned cloudy and was refluxed overnight. The mixture was cooled to RTand ethyl formate (4.66 mL, 58 mmol) added dropwise. The suspension wasstirred overnight, turning bright yellow. The R.M. was transferred to a2000-mL sep. funnel with ether (50 mL), and washed with 10% sulphuricacid (200 mL), water (2×200 mL) and brine (200 mL). The organic wasdried over anhydrous MgSO₄, filtered and concentrated. Crude yield was14.5 g. TLC indicated that majority of product was the methyl formate,which was purified by column chromatography. The purified formate wastransferred to a 1000 mL round bottom flask and EtOH (600 mL) and astirbar added. With stirring, water (25 mL-forming ˜5% aqueous solution)was slowly added, followed by KOH (2.0 g, 35.7 mmol). After 1 hour,solution had turned pale yellow. TLC indicated reaction had gone tocompletion. The solution was concentrated by rotovap to 50% of itsvolume and then poured into 200 mL of 5% HCl. The aqueous phase wasextracted with ether (3×200 mL). The ether fractions were combined andwashed with water (3×200 mL), dried (MgSO₄) and concentrated to yield8.8 g of di-γ-linolenyl methanol (16.8 mmol, 58%).

Synthesis of γ-LenMC3 (Compound 24)

Di-γ-linolenyl methanol (Compound 23) (2.5 g, 4.76 mmol),dimethylaminobutyric acid hydrochloride (970 mg, 5.77 mmol) and a stirbar were added to 100 mL RBF. The flask was flushed with nitrogen andanhydrous DCM (40 mL) added, followed by EDCI (1.2 g, 6.26 mmol), DIPEA(2.1 mL, 12.1 mmol) and DMAP (150 mg, 1.23 mmol). The reaction wasstirred overnight. The reaction was diluted with DCM (100 mL) and washedwith sat. NaHCO₃ (100 mL), water (200 mL) and sat. NaCL (100 mL).Aqueous washes were combined and extracted with DCM (2×50 mL). Organicswere then combined, dried (MgSO₄) and concentrated to yield a yellowoil. This was purified by chromatography to yield γ-Len-MC3 as a paleyellow oil (2.6 g, 4.1 mmol, 86%).

Synthesis of CP-γ-LenMC3 (Compound 25)

To a 250 mL RBF was added γ-LenMC3 (Compound 24) (1.28 g, 2.0 mmol), astirbar and anhydrous DCM (40 mL). The flask was flushed with N₂ andcooled to 0° C., then a 1M solution of diethylzinc in hexanes added (30mL, 30 mmol, ˜5 equivalents per olefin). The solution was stirred for 1hour at 0° C., then diiodomethane (2.4 mL 50 mmol) added and thereaction stirred overnight at RT. The reaction mixture was concentratedand then redissolved in EtOAc (50 mL). The EtOAc was washed successivelywith 5% HCl (2×50 mL), water (50 mL), NaHCO₃ (50 mL), water (50 mL), andbrine (50 mL). The aqueous washes were combined and extracted with DCM(2×50 mL). All organics were combined, dried and concentrated to yieldcrude CP-γ-LenMC3. ¹H-NMR indicated some olefins still to be present, sothe compound was treated again, using the same procedures and amountsoutlined above. This time ¹H-NMR indicated total conversion of theolefins. Final yield after chromatography was 1.3 g, 1.8 mmol, 90%.

Example 12. Synthesis of MC3

MC3 (Compound 26) having the structure shown below was synthesized asdescribed in Scheme 9 below

STEP 1: Magnesium bromide etherate (34 g, 110 mmol) and a stir bar wereadded to a 2000 mL round bottom flask. The flask was sealed and flushedwith nitrogen. Anhydrous diethyl ether (400 mL) was added via canulla. Asolution of linolenyl mesylate (20 g, 58 mmol) in anhydrous ether (300mL) was then added, and the suspension stirred overnight. The suspensionwas poured into 500 mL of chilled water and transferred to a 2000-mLseparating funnel. After shaking, the organic phase was separated. Theaqueous phase was then extracted with ether (2×250 mL) and all etherphases combined. The ether phase was washed with water (2×250 mL), brine(250 mL) and dried over anhydrous Mg₂SO₄. The solution was filtered,concentrated and purified by flash chromatography. Final yield 18.9 g,99%.

STEP 2: A 1 liter RBF was charged with magnesium turnings (11.1 g, 463mmol), anhydrous THF (65 mL) and stir-bar and flushed with nitrogen. Ina separate flask, a solution of linoleyl bromide (140 g, 425 mL) inanhydrous THF (150 mL) was prepared, and 20 mL of this solution added tothe magnesium. When most of the heat had dissipated, the remainder ofthe bromide was added over a period of 15 minutes. Temperature was thenmaintained at 45° C. for 4 h. The reaction was then cooled (0° C.).Using a dropping funnel, a solution of ethyl formate (32.4 g, 438 mmol)in anhydrous THF (150 mL) was added over a 40 minute period. Thereaction was stirred overnight at room temperature. The reaction wascooled to −15° C. and 5N HCl (185 mL) added slowly. The mixture wastransferred to a 2 L separating funnel separated. Water (150 mL) andhexane (150 mL) were added, the mixture washed, and again the aqueousremoved. The organic was washed a final time with water (150 mL) andthen concentrated to a yellow oil. The yellow oil was stirred with amixture of EtOH (210 mL), water (30 mL) and KOH (15.8 g) for 1.5 h atroom temp. The EtOH was evaporated and the residue treated with hexane(50 mL). 5N HCl (200 mL) was added via dropping funnel. The organic waswashed with water (2×50 mL) evaporated, dried and purified bychromatography (0-5% EtOAc in hexane, 91 g, 81%).

STEP 3: Dilinoleylmethanol (7.8 g, 14.9 mmol), dimethylaminobutyric acidhydrochloride (2.99 g, 17.8 mmol) and a stir bar were added to 500 mLRBF. The flask was flushed with nitrogen and anh. DCM (120 mL) added,followed by EDCI (3.6 g, 18.8 mmol), DIPEA (6.3 mL, 36.3 mmol) and DMAP(450 mg, 3.69 mmol). The reaction was stirred overnight. The reactionwas diluted with DCM (300 mL) and washed with sat. NaHCO₃ (200 mL),water (300 mL) and sat. NaCL (200 mL). Each aq. wash was extracted oncewith DCM (50 mL). Organics were combined, dried (MgSO₄) and concentratedto yield a yellow oil with some crystalline matter. This was purified bychromatography (0-2% MeOH in CHCl₃) to yield Lin-MC3 as a pale yellowoil (9.0 g, 14.1 mmol, 95%).

Example 13. Synthesis of CP-MC3

CP-MC3 (Compound 27) having the structure shown below was synthesized asdescribed in Scheme 10 below.

To a solution of MC3 (2.1 g, 3.27 mmol) in anhydrous CH₂Cl₂ (60 mL) at0° C. under nitrogen was added 2.5 equivalents diethyl zinc (1M solutionin hexanes) (31 mL, 3 mmol). The solution was stirred for 1 hour andthen 2.5 equivalents diiodomethane (2.5 mL, 31 mmol) added. The reactionwas stirred overnight at room temp. The white suspension was poured intoice (50 mL) and diluted to 200 mL using ethyl acetate (white soliddissolved). 5% HCl (100 mL) was added to wash. The aqueous (acidic)layer was removed and extracted with ethyl acetate (2×125 mL). Theorganic (top) layer was washed again with 5% HCl, then saturated NaHCO₃,water, and brine (150 mL each), dried on MgSO₄, filtered, andconcentrated to yield a cloudy pale yellow oil. The procedure above wasrepeated once to ensure 100% cyclopropylation. Product was a pale yellowoil. The oil was purified by column chromatography eluting with CHCl₃ toafford a pale yellow oil. Final yield 1.11 g, 51%.

Example 14. Lipid Encapsulation of siRNA

All siRNA molecules used in these studies were chemically synthesizedand annealed using standard procedures.

In some embodiments, siRNA molecules were encapsulated into serum-stablenucleic acid-lipid particles (SNALP) composed of the following lipids:(1) the lipid conjugate PEG2000-C-DMA (3-N-[(-methoxypoly(ethyleneglycol)2000)carbamoyl]-1,2-dimyristyloxypropylamine); (2) one or morecationic lipids or salts thereof (e.g., cationic lipids of Formula I-IIIof the invention and/or other cationic lipids described herein); (3) thephospholipid DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) (AvantiPolar Lipids; Alabaster, Ala.); and (4) synthetic cholesterol(Sigma-Aldrich Corp.; St. Louis, Mo.) in the molar ratio1.4:57.1:7.1:34.3, respectively. In other words, siRNA molecules wereencapsulated into SNALP of the following “1:57” formulation: 1.4%PEG2000-C-DMA; 57.1% cationic lipid; 7.1% DPPC; and 34.3% cholesterol.It should be understood that the 1:57 formulation is a targetformulation, and that the amount of lipid (both cationic andnon-cationic) present and the amount of lipid conjugate present in theformulation may vary. Typically, in the 1:57 formulation, the amount ofcationic lipid will be 57.1 mol %±5 mol %, and the amount of lipidconjugate will be 1.4 mol %±0.5 mol %, with the balance of the 1:57formulation being made up of non-cationic lipid (e.g., phospholipid,cholesterol, or a mixture of the two).

In other embodiments, siRNA were encapsulated into SNALP composed of thefollowing lipids: (1) the lipid conjugate PEG750-C-DMA(3-N-[(-Methoxypoly(ethyleneglycol)750)carbamoyl]-1,2-dimyristyloxypropylamine); (2) one or morecationic lipids or salts thereof (e.g., cationic lipids of Formula I-IIIof the invention and/or other cationic lipids described herein); (3) thephospholipid DPPC; and (4) synthetic cholesterol in the molar ratio6.76:54.06:6.75:32.43, respectively. In other words, siRNA wereencapsulated into SNALP of the following “7:54” formulation: 6.76 mol %PEG750-C-DMA; 54.06 mol % cationic lipid; 6.75 mol % DPPC; and 32.43 mol% cholesterol. Typically, in the 7:54 formulation, the amount ofcationic lipid will be 54.06 mol %±5 mol %, and the amount of lipidconjugate will be 6.76 mol %±1 mol %, with the balance of the 7:54formulation being made up of non-cationic lipid (e.g., phospholipid,cholesterol, or a mixture of the two).

For vehicle controls, empty particles with identical lipid compositionmay be formed in the absence of siRNA.

Example 15. pK_(a) Measurements of SNALP Formulations Containing NovelCyclic Cationic Lipids

This example demonstrates the determination of pK_(a) values of various1:57 SNALP formulations containing novel cyclic cationic lipidsdescribed herein with an siRNA targeting apolipoprotein B (ApoB).

1:57 SNALP formulations containing encapsulated ApoB siRNA were preparedas described in Section VI above with the following cationic lipids: (1)DLinDMA; (2) CP-DLinDMA; (3) CP-DLenDMA; (4) CP-γ-DLenDMA(“CP-g-DLenDMA”); (5) CP-DODMA; (6) CP-DPetroDMA; (7) C2-TLinDMA; (8)γ-LenMC3 (“g-Len-MC3”); (9) CP-γ-LenMC3 (“CP-g-Len-MC3”); (10) LenMC3;and (11) CP-LenMC3.

The apparent pK_(a) of the cationic lipids present in these SNALPformulations was determined using a2-(p-toluidinyl)-naphthalene-6-sodium sulfonate (TNS) assay. TNS is anegatively-charged indicator of membrane potential that iselectrostatically attracted to positively charged membranes (see, Baileyand Cullis, Biochemistry, 33 12573-80 (1994)). Subsequent adsorption tothe lipid membrane results in the immediate environment of the TNSbecoming more lipophilic, removing the water molecules that otherwisequench TNS fluorescence. As a result, TNS measures the surface potentialof the particle, wherein the more positive the surface potential, thegreater the level of fluorescence. The surface pK_(a) values of eachSNALP formulation were determined by varying the local pH in thepresence of TNS. By plotting fluorescence versus pH, the pK_(a) of thecationic lipid can be estimated in the particle as the pH wherefluorescence equals 50% of total fluorescence.

FIGS. 1-2 show the results of the TNS assays, wherein the 1:57 SNALPhave the following pK_(a) values: (1) DLinDMA ˜5.8; (2) CP-DLinDMA ˜5.8;(3) CP-DLenDMA ˜5.85; (4) CP-g-DLenDMA ˜5.8; (5) CP-DODMA ˜5.8; (6)CP-DPetroDMA ˜5.95; (7) C2-TLinDMA ˜5.85; (8) g-Len-MC3 ˜6.1; (9)CP-g-Len-MC3 ˜6.0; (10) LenMC3 ˜6.1; and (11) CP-LenMC3 ˜6.1.

Example 16. Characterization of SNALP Formulations Containing NovelCyclic Cationic Lipids

This example demonstrates the efficacy of 1:57 SNALP formulationscontaining various novel cyclic cationic lipids of Formula I describedherein with an siRNA targeting ApoB in a mouse liver model. The ApoBsiRNA sequence used in this study is provided in Table 1.

TABLE 1 % 2′OMe- % Modified siRNA ApoB siRNA Sequence Modifiedin DS Region ApoB-10164 5′-AGU G UCA U CACAC U GAAUACC-3′ 7/42 = 16.7%7/38 = 18.4% (SEQ ID NO: 1) 3′-GU U CACAGUAGU G U G AC U UAU-5′(SEQ ID NO: 2) Column 1: The number after ″ApoB″ refers to thenucleotide position of the 5′ base of the sense strand relative to thehuman ApoB mRNA sequence NM_000384. Column 2: 2′OMe nucleotides areindicated in bold and underlined. The 3′-overhangs on one or bothstrands of the siRNA molecule may alternatively comprise 1-4deoxythymidine (dT) nucleotides, 1-4 modified and/or unmodified uridine(U) ribonucleotides, or 1-2 additional ribonucleotides havingcomplementarity to the target sequence or the complementary strandthereof. Column 3: The number and percentage of 2′OMe-modifiednucleotides in the siRNA molecule are provided. Column 4: The number andpercentage of modified nucleotides in the double-stranded (DS) region ofthe siRNA molecule are provided.

1:57 SNALP formulations containing encapsulated ApoB siRNA were preparedas described in Section VI above with the following cationic lipids: (1)DLinDMA; (2) CP-DLinDMA; (3) CP-DLenDMA; (4) CP-γ-DLenDMA(“CP-g-DLenDMA”); (5) CP-DODMA; (6) CP-DPetroDMA; and (7) C2-TLinDMA.Table 2 provides exemplary features of these SNALP formulations,including particle size, polydispersity, and percent encapsulation.

TABLE 2 Size (nm) Poly Encapsulation % 1:57 DLinDMA 76.11 0.045 81 1:57CP-DODMA 77.77 0.034 87 1:57 CP-DPetroDMA 75.83 0.035 90 1:57 CP-DLinDMA72.39 0.028 90 1:57 CP-DLenDMA 75.82 0.024 89 1:57 CP-g-DLenDMA 68.150.062 83

Each SNALP formulation (6:1 L:D) was administered by intravenous (IV)injection at 0.1 mg/kg into female Balb/c mice (n=3 per group). LiverApoB mRNA levels were evaluated at 48 hours after SNALP administrationby a branched DNA assay (QuantiGene assay) to assess ApoB mRNA relativeto the housekeeping gene GAPDH.

FIG. 3 shows a comparison of the liver ApoB mRNA knockdown activity ofeach of these SNALP formulations (Error bars=SD). In particular, FIG. 3shows that a SNALP formulation containing either CP-DLinDMA, CP-DLenDMA,or CP-γ-DLenDMA displayed similar or greater ApoB silencing activitycompared to a SNALP formulation containing the DLinDMA benchmarkcationic lipid. Notably, SNALP formulations containing the novel cycliccationic lipids of Formula I displayed improved stability over SNALPformulations containing their polyunsaturated counterparts whenevaluated under various different storage conditions using techniquessuch as, e.g., analysis of any change in particle size over time and/ordetermination of any degradation of the nucleic acid payload and/orcationic lipid.

Example 17. Characterization of Additional SNALP Formulations ContainingNovel Cyclic Cationic Lipids

This example demonstrates the efficacy of 1:57 SNALP formulationscontaining various novel cyclic cationic lipids of Formula II describedherein with an siRNA targeting ApoB in a mouse liver model. The ApoBsiRNA sequence used in this study is provided in Table 1 above.

1:57 SNALP formulations containing encapsulated ApoB siRNA were preparedas described in Section VI above with the following cationic lipids: (1)DLin-C2K-DMA (“C2K”); (2) DLin-M-C3-DMA (“MC3”); (3) LenMC3(“DLen-MC3”); (4) CP-LenMC3 (“CP-DLen-MC3”); (5) D-γ-Len-C2K-DMA(“g-DLen-C2K-DMA”); (6) CP-D-γ-Len-C2K-DMA (“CP-g-DLen-C2K-DMA”); (7)DLen-C2K-DMA; and (8) CP-DLen-C2K-DMA.

For dose response studies, SNALP formulations were administered by IVinjection at 0.01 mg/kg, 0.033 mg/kg, or 0.1 mg/kg into female Balb/cmice (n=3 per group). Liver ApoB mRNA levels were evaluated at 48 hoursafter SNALP administration by a branched DNA assay (QuantiGene assay) toassess ApoB mRNA relative to the housekeeping gene GAPDH.

FIG. 4 shows a comparison of the liver ApoB mRNA knockdown activity ofeach of these SNALP formulations at three different doses (Errorbars=SD), as well as the KD50 values obtained for each of theseformulations. In particular, FIG. 4 shows that a SNALP formulationcontaining CP-g-DLen-C2K-DMA displayed similar ApoB silencing activityat higher doses and similar KD50 value compared to a SNALP formulationcontaining the C2K benchmark cationic lipid. Furthermore, FIG. 4 showsthat a SNALP formulation containing CP-DLen-C2K-DMA displayedconsiderable potency in silencing ApoB mRNA expression. Notably, SNALPformulations containing the novel cyclic cationic lipids of Formula IIdisplayed improved stability over SNALP formulations containing theirpolyunsaturated counterparts when evaluated under various differentstorage conditions using techniques such as, e.g., analysis of anychange in particle size over time and/or determination of anydegradation of the nucleic acid payload and/or cationic lipid.

Example 18. Characterization of Additional SNALP Formulations ContainingNovel Cyclic Cationic Lipids

This example demonstrates the efficacy of 1:57 SNALP formulationscontaining various novel cyclic cationic lipids of Formula III describedherein with an siRNA targeting ApoB in a mouse liver model. The ApoBsiRNA sequence used in this study is provided in Table 1 above.

1:57 SNALP formulations containing encapsulated ApoB siRNA were preparedas described in Section VI above with the following cationic lipids: (1)DLin-C2K-DMA (“C2K”); (2) DLin-M-C3-DMA (“MC3”); (3) γ-LenMC3(“g-Len-MC3”); (4) CP-γ-LenMC3 (“CP-g-Len-MC3”); (5) LenMC3; and (6)CP-LenMC3. Table 3 provides exemplary features of these SNALPformulations, including particle size, polydispersity, and percentencapsulation.

TABLE 3 Initial Final Total Size Size siRNA Formulation (nm) (nm) PolyEncaps (mg/ml) 1:57 C2K 80 88 0.030 98% 5.2 1:57 MC3 80 84 0.034 99% 4.61:57 g-Len-MC3 76 79 0.052 98% 4.8 1:57 CP-g-Len-MC3 79 82 0.037 100% 5.1 1:57 Len-MC3 79 84 0.049 98% 5.8 1:57 CP-Len-MC3 76 83 0.029 100% 5.6

For dose response studies, SNALP formulations were administered by IVinjection at 0.01 mg/kg, 0.033 mg/kg, or 0.1 mg/kg into female Balb/cmice (n=3 per group). Liver ApoB mRNA levels were evaluated at 48 hoursafter SNALP administration by a branched DNA assay (QuantiGene assay) toassess ApoB mRNA relative to the housekeeping gene GAPDH.

FIG. 5 shows a comparison of the liver ApoB mRNA knockdown activity ofeach of these SNALP formulations at three different doses (Errorbars=SD). FIG. 6 shows the KD50 calculation and values obtained for eachof these SNALP formulations. In particular, FIG. 5 shows that a SNALPformulation containing either CP-γ-LenMC3 or CP-LenMC3 displayed similarApoB silencing activity compared to a SNALP formulation containing theC2K benchmark cationic lipid at all three doses. FIG. 6 shows that aSNALP formulation containing either CP-γ-LenMC3 or CP-LenMC3 displayedsimilar KD50 values compared to a SNALP formulation containing the C2Kbenchmark cationic lipid. Notably, SNALP formulations containing thenovel cyclic cationic lipids of Formula III displayed improved stabilityover SNALP formulations containing their polyunsaturated counterpartswhen evaluated under various different storage conditions usingtechniques such as, e.g., analysis of any change in particle size overtime and/or determination of any degradation of the nucleic acid payloadand/or cationic lipid.

Example 19. Characterization of Additional SNALP Formulations ContainingNovel Cyclic Cationic Lipids

This example demonstrates the efficacy of 1:57 SNALP formulationscontaining various novel cyclic cationic lipids of Formula II-IIIdescribed herein with an siRNA targeting ApoB in a mouse liver model.The ApoB siRNA sequence used in this study is provided in Table 1 above.

1:57 SNALP formulations containing encapsulated ApoB siRNA were preparedas described in Section VI above with the following cationic lipids: (1)DLin-C2K-DMA (“C2K”); (2) MC2MC; (3) MC3 Ether; (4) Pan-MC3; (5) CP-MC3;and (6) CP-C2K. SNALP formulations were administered by IV injection at0.05 mg/kg into female Balb/c mice (n=3 per group). Liver ApoB mRNAlevels were evaluated at 48 hours after SNALP administration by abranched DNA assay (QuantiGene assay) to assess ApoB mRNA relative tothe housekeeping gene GAPDH.

FIG. 7 shows a comparison of the liver ApoB mRNA knockdown activity ofeach of these SNALP formulations (Error bars=SD). In particular, FIG. 7shows that a SNALP formulation containing either CP-MC3 or CP-C2Kdisplayed similar ApoB silencing activity compared to a SNALPformulation containing the C2K benchmark cationic lipid. Notably, SNALPformulations containing the novel cyclic cationic lipids of FormulaII-III displayed improved stability over SNALP formulations containingtheir polyunsaturated counterparts when evaluated under variousdifferent storage conditions using techniques such as, e.g., analysis ofany change in particle size over time and/or determination of anydegradation of the nucleic acid payload and/or cationic lipid.

It is to be understood that the above description is intended to beillustrative and not restrictive. Many embodiments will be apparent tothose of skill in the art upon reading the above description. The scopeof the invention should, therefore, be determined not with reference tothe above description, but should instead be determined with referenceto the appended claims, along with the full scope of equivalents towhich such claims are entitled. The disclosures of all articles andreferences, including patent applications, patents, PCT publications,and Genbank Accession Nos., are incorporated herein by reference for allpurposes.

1. A cationic lipid of Formula III having the following structure:

or salts thereof, wherein: R¹ and R² are either the same or differentand are independently hydrogen (H) or an optionally substituted C₁-C₆alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl, or R¹ and R² may join to form anoptionally substituted heterocyclic ring; R³ is either absent or ishydrogen (H) or a C₁-C₆ alkyl to provide a quaternary amine; R⁴ and R⁵are either the same or different and are independently an optionallysubstituted C₁₀-C₂₄ alkyl, C₁₀-C₂₄ alkenyl, C₁₀-C₂₄ alkynyl, or C₁₀-C₂₄acyl, wherein at least one of R⁴ and R⁵ comprises at least oneoptionally substituted cyclic alkyl group; X is O, S, N(R⁶), C(O),C(O)O, OC(O), C(O)N(R⁶), N(R⁶)C(O), OC(O)N(R⁶), N(R⁶)C(O)O, C(O)S,C(S)O, S(O), S(O)(O), C(S), or an optionally substituted heterocyclicring, wherein R⁶ is hydrogen (H) or an optionally substituted C₁-C₁₀alkyl, C₂-C₁₀ alkenyl, or C₂-C₁₀ alkynyl; and Y is either absent or isan optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl.2-16. (canceled)
 17. A cationic lipid of Formula I having the followingstructure:

or salts thereof, wherein: R¹ and R² are either the same or differentand are independently hydrogen (H) or an optionally substituted C₁-C₆alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl, or R¹ and R² may join to form anoptionally substituted heterocyclic ring; R³ is either absent or ishydrogen (H) or a C₁-C₆ alkyl to provide a quaternary amine; R⁴ and R⁵are either the same or different and are independently an optionallysubstituted C₁₀-C₂₄ alkyl, C₁₀-C₂₄ alkenyl, C₁₀-C₂₄ alkynyl, or C₁₀-C₂₄acyl, wherein at least one of R⁴ and R⁵ comprises at least oneoptionally substituted cyclic alkyl group; X and Y are either the sameor different and are independently O, S, N(R⁶), C(O), C(O)O, OC(O),C(O)N(R⁶), N(R⁶)C(O), OC(O)N(R⁶), N(R⁶)C(O)O, C(O)S, C(S)O, S(O),S(O)(O), C(S), or an optionally substituted heterocyclic ring, whereinR⁶ is hydrogen (H) or an optionally substituted C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, or C₂-C₁₀ alkynyl; and Z is either absent or is an optionallysubstituted C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl. 18-33.(canceled)
 34. A cationic lipid of Formula II having the followingstructure:

or salts thereof, wherein: R¹ and R² are either the same or differentand are independently an optionally substituted C₁-C₆ alkyl, C₂-C₆alkenyl, or C₂-C₆ alkynyl, or R¹ and R² may join to form an optionallysubstituted heterocyclic ring; R³ is either absent or is hydrogen (H) ora C₁-C₆ alkyl to provide a quaternary amine; R⁴ and R⁵ are either thesame or different and are independently an optionally substitutedC₁₀-C₂₄ alkyl, C₁₀-C₂₄ alkenyl, C₁₀-C₂₄ alkynyl, or C₁₀-C₂₄ acyl,wherein at least one of R⁴ and R⁵ comprises at least one optionallysubstituted cyclic alkyl group; m, n, and p are either the same ordifferent and are independently either 0, 1, or 2, with the proviso thatm, n, and p are not simultaneously 0; X and Y are either the same ordifferent and are independently O, S, N(R⁶), C(O), C(O)O, OC(O),C(O)N(R⁶), N(R⁶)C(O), OC(O)N(R⁶), N(R⁶)C(O)O, C(O)S, C(S)O, S(O),S(O)(O), C(S), wherein R⁶ is hydrogen (H) or an optionally substitutedC₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, or C₂-C₁₀ alkynyl; and Z is either absentor is an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆alkynyl. 35-49. (canceled)
 50. A lipid particle comprising a cationiclipid of claim
 1. 51-57. (canceled)
 58. The lipid particle of claim 50,wherein the particle further comprises a therapeutic agent. 59-66.(canceled)
 67. A pharmaceutical composition comprising a lipid particleof claim 50, and a pharmaceutically acceptable carrier.
 68. A method forintroducing a therapeutic agent into a cell, the method comprising:contacting the cell with a lipid particle of claim
 58. 69. (canceled)70. A method for the in vivo delivery of a therapeutic agent, the methodcomprising: administering to a mammal a lipid particle of claim 58.71-72. (canceled)
 73. A method for treating a disease or disorder in amammal in need thereof, the method comprising: administering to themammal a therapeutically effective amount of a lipid particle of claim58. 74-75. (canceled)
 76. A lipid particle comprising a cationic lipidof claim
 17. 77. The lipid particle of claim 76, wherein the particlefurther comprises a therapeutic agent.
 78. A pharmaceutical compositioncomprising a lipid particle of claim 76, and a pharmaceuticallyacceptable carrier.
 79. A method for introducing a therapeutic agentinto a cell, the method comprising contacting the cell with a lipidparticle of claim
 77. 80. A method for the in vivo delivery of atherapeutic agent, the method comprising administering to a mammal alipid particle of claim
 77. 81. A method for treating a disease ordisorder in a mammal in need thereof, the method comprisingadministering to the mammal a therapeutically effective amount of alipid particle of claim
 77. 82. A lipid particle comprising a cationiclipid of claim
 34. 83. The lipid particle of claim 82, wherein theparticle further comprises a therapeutic agent.
 84. A pharmaceuticalcomposition comprising a lipid particle of claim 82, and apharmaceutically acceptable carrier.
 85. A method for introducing atherapeutic agent into a cell, the method comprising contacting the cellwith a lipid particle of claim
 83. 86. A method for the in vivo deliveryof a therapeutic agent, the method comprising administering to a mammala lipid particle of claim
 83. 87. A method for treating a disease ordisorder in a mammal in need thereof, the method comprisingadministering to the mammal a therapeutically effective amount of alipid particle of claim 83.